KR20240013787A - Plasma fractionation by sequential extraction - Google Patents

Abstract

The present invention relates to a method for purifying immunoglobulins and/or albumin from a plasma sample, the method comprising mixing the plasma sample with medium chain fatty acids and recovering soluble immunoglobulins from the mixture.

Description

Plasma fractionation by sequential extraction

Technology field

The present invention relates to methods and systems for extracting immunoglobulins (Igs), such as immunoglobulin G (IgG), from plasma.

Cross-references to previous applications

This application claims priority to Australian Provisional Patent Application No. 2021901577, the entire contents of which are incorporated herein by reference.

The demand for purified proteins, such as specific antibodies, has increased significantly. These purified proteins can be used for therapeutic and/or diagnostic purposes.

Human plasma contains well-established and recognized plasma protein products, such as human albumin (HSA), immunoglobulins (IgG), coagulation factor remnants (coagulation factor VIII, coagulation factor IX, prothrombin complex, etc.) and inhibitors (antithrombin). , C1 inhibitors, etc.) have been utilized industrially for decades. In the process of developing such plasma-derived drugs, plasma fractionation methods are established to generate intermediate products enriched in specific protein fractions, which are used as starting compositions of plasma-protein product/s. The general process is reviewed, for example, in Molecular Biology of Human Proteins (Schultze H.E., Heremans J.F.; Volume I: Nature and Metabolism of Extracellular Proteins 1966, Elsevier Publishing Company; p. 236-317). This type of separation technology allows the production of multiple therapeutic plasma-protein products from the same pool of plasma donors. This has been adopted as the industry standard for plasma fractionation because it is economically more advantageous than producing a single plasma-protein product from a single donor pool.

One example of this type of fractionation process, cold ethanol fractionation of plasma, was pioneered by E.J Cohn and his team during World War II, primarily for albumin purification (Cohn EJ, et al . 1946, J. Am. Chem. Soc. 62: 459-475). The Cohn fractionation process involves gradually increasing the ethanol concentration from 0% to 40% while lowering the pH from neutral (pH 7) to about 4.8 to precipitate albumin. Although cone fractionation has evolved over the past 70 years, most commercial plasma fractionation processes are based on the original process or its variants (e.g. Kistler/Nitschmann) and are based on pH, ionic strength, and , utilizing differences in solvent polarity and alcohol concentration to separate plasma into a series of major precipitated protein fractions (e.g., Cohn's fractions I to V).

Various cone fractionation processes have been developed for the purpose of improving multivalent IgG recovery. For example, Oncley and colleagues generated activated immunoglobulin serum fractions using Cohn fractions II+III as starting material with different combinations of cold ethanol, pH, temperature, and protein concentration than those described by Cohn. (Oncley et al., (1949) J. Am. Chem. Soc. 71, 541-550). Today, the Onclay method is the classic method used for multivalent IgG production. Nevertheless, it is known that approximately 5% of gammaglobulins (antibody-rich fraction) are co-precipitated with fraction I, and approximately 15% of the total gammaglobulins present in plasma are lost in fractions II+III stages (ref. [Table III, Cohn EJ, et al. 1946, J. Am. Chem. Soc. 62: 459-475]). The Kistler/Nitzmann method aimed to improve IgG recovery by reducing the ethanol content in some precipitation steps (precipitation B vs fraction III). However, increased yield comes at the expense of purity (Kistler & Nitschmann, (1962) Vox Sang. 7, 414-424).

Initially, immunoglobulin G (IgG) products extracted from these fractionation processes were successfully used to prevent and treat various infectious diseases. However, because ethanol fractionation is a relatively crude process, the IgG product contains enough impurities and aggregates to allow for intramuscular administration only. Subsequently, further improvements in the purification process resulted in IgG preparations suitable for intravenous administration (referred to as IVIg) and subcutaneous administration (referred to as SCIg).

It is estimated that approximately 30 million liters of plasma were processed worldwide in 2010, providing a variety of therapeutic products, including approximately 500 tonnes of albumin and 100 tonnes of IVIg. The IVIg market accounts for approximately 40 to 50% of the total plasma fraction market (P. Robert, Worldwide supply and demand of plasma and plasma derived medicines (2011) J. Blood and Cancer, 3, 111-120). Therefore, since the demand for IVIg remains strong (along with the increasing demand for SCIg), there still exists a need to improve immunoglobulin recovery from plasma and related fractions. Preferably, this should be achieved in a way that ensures that the recovery of other plasma derived therapeutic proteins is not adversely affected.

From a commercial standpoint, the initial fractionation process is critical to the overall production time and costs associated with the production of therapeutic proteins, especially plasma-derived proteins, as subsequent purification steps determine the yield and This is because it varies depending on purity. To improve protein yield at lower operating costs, several variations of the cold ethanol fractionation process have been developed for plasma-derived proteins, but generally higher protein yields result in lower purity.

There is a need for improved methods and systems for producing proteins on an industrial scale from blood-derived plasma or serum and that meet stringent safety standards. The downstream technologies currently used are relatively expensive, and their yields are suboptimal. Therefore, it is important to develop more efficient and economical methods for extracting and purifying proteins, such as immunoglobulins, from plasma.

References herein to prior art mean that such prior art forms part of the general general knowledge in any jurisdiction, or that such prior art could reasonably be expected to be understood and relevant, and/or that other prior art may be recognized by a person skilled in the art. It does not acknowledge or suggest that it can be combined with .

The present invention is based on the discovery that relatively pure immunoglobulin, especially IgG preparations can be obtained directly from plasma without the need for an ethanol fractionation step.

Furthermore, the present invention is based on the discovery that relatively pure immunoglobulin, especially IgG and albumin preparations can be obtained directly from plasma without the need for an ethanol fractionation step.

Furthermore, the present invention is based on the discovery that relatively pure albumin preparations can be obtained directly from plasma without the need for an ethanol fractionation step.

Accordingly, in a first aspect, the present invention provides a method for obtaining an immunoglobulin solution, comprising:

- contacting the blood-derived plasma sample with medium chain fatty acids under conditions capable of selectively precipitating albumin from the blood-derived plasma sample,

- the conditions are a pH range of about 4.6 to about 5.0,

- providing for obtaining an immunoglobulin solution, wherein the immunoglobulin solution is formed by the contacting step,

Preferably, the conditions also include a conductivity of about 5 to about 12 mS/cm, preferably 8 to about 12 mS/cm.

In a second aspect, the present invention provides a method for purifying immunoglobulins, comprising:

- contacting the blood-derived plasma sample with medium chain fatty acids under conditions of a pH ranging from about 4.6 to about 5.0 to allow albumin to selectively precipitate from the blood-derived plasma sample,

- thereby forming a suspension comprising a soluble protein-containing component comprising immunoglobulins and an insoluble protein-containing component comprising albumin, and

- separating the soluble protein-containing component from the insoluble protein-containing component to obtain a purified immunoglobulin solution.

The step of separating the soluble protein-containing component from the insoluble protein-containing component preferably includes subjecting the suspension to sequential extractive filtration. Preferably, separating the soluble protein-containing component from the insoluble protein-containing component comprises:

- feeding the suspension to a filtration unit comprising a dynamic filter element configured to produce a first retentate and a first filtrate, and

- Including recovering the first filtrate.

The first retentate comprising the insoluble protein-containing component is generally rich in albumin, and the first filtrate comprising the soluble protein-containing component is rich in immunoglobulins.

The first filtrate enriched in immunoglobulins may be subjected to a concentration step prior to further processing. The concentration step may include a continuous concentration process, whereby the first filtrate is fed to a second filtration unit, said second filtration unit being configured to filter a second immunoglobulin-enriched retentate and a second immunoglobulin-depleted retentate. and a cross-flow filter element or TFF configured to produce liquid. Optionally, a second filtrate depleted in immunoglobulins can be streamed back to the first tank and/or a second retentate enriched in immunoglobulins can be streamed back to the second tank.

As further described herein, it will be appreciated that the immunoglobulin-enriched solution may be further purified using standard techniques.

In a preferred embodiment, a method for purifying immunoglobulins, comprising:

- To allow selective precipitation of albumin from the blood-derived plasma sample, the blood-derived plasma sample and medium chain fatty acids are subjected to a pH range of about 4.6 to about 5.0 and a conductivity of about 8 to about 12 mS/cm. contacting step,

- thereby forming a suspension comprising a soluble protein-containing component comprising immunoglobulins and an insoluble protein-containing component comprising albumin, and

- separating the soluble protein-containing component from the insoluble protein-containing component to obtain a purified immunoglobulin solution,

- Optionally, the step of separating the soluble protein-containing component from the insoluble protein-containing component comprises feeding the suspension to a filtration unit comprising a dynamic filter element configured to produce a first retentate and a first filtrate, and 1 A method for purifying immunoglobulin is provided, comprising recovering the filtrate.

If the pH needs to be further adjusted, the pH of the blood-derived plasma sample can be adjusted without substantially diluting the sample, for example, with concentrated acid (e.g., acetic acid) or acid and base (e.g., NaOH). ) can be adjusted directly by adding a combination of Likewise, the conductivity of blood-derived plasma can be adjusted directly without substantially diluting the sample.

In a third aspect, there is provided a method for purifying immunoglobulins from plasma, comprising:

a) In a first tank, the blood-derived plasma sample is placed under conditions of a pH ranging from about 4.6 to about 5.0 and optionally a conductivity of from about 8 to about 12 mS/cm to allow selective precipitation of albumin from the blood-derived plasma sample. mixing the derived plasma sample and medium chain fatty acids to form a suspension comprising soluble immunoglobulins and insoluble albumin;

b) feeding the suspension to a first filtration unit, wherein the first filtration unit is configured to produce a first retentate comprising the insoluble albumin and a first filtrate comprising the soluble immunoglobulin. The supply step, including,

c) optionally streaming the first residue into the first tank to dilute the suspension in the first tank,

d) recovering the first filtrate from a second tank, and

A method is provided for purifying immunoglobulins from plasma, comprising the step of e) optionally concentrating said first filtrate.

Accordingly, in a fourth aspect, the present invention provides a method for obtaining an immunoglobulin solution and an albumin precipitate, comprising:

- contacting the blood-derived plasma sample with medium chain fatty acids under conditions allowing albumin to selectively precipitate from the blood-derived plasma sample,

- the conditions are a pH range of about 4.2 to about 5.0,

- A method for obtaining an immunoglobulin solution and an albumin precipitate is provided, whereby an immunoglobulin solution and an albumin precipitate are formed.

Preferably, the conditions are also such that the conductivity is from about 5 to about 12 mS/cm, preferably from 8 to about 12 mS/cm. More preferably, when the blood-derived plasma is diluted, the conductivity is greater than 5 mS/cm and less than 12 mS/cm or less than about 12 mS/cm. When blood-derived plasma is not diluted, the conductivity is greater than or equal to 8 mS/cm but less than 12 mS/cm or less than about 12 mS/cm.

In a fifth aspect, the present invention provides a method for purifying immunoglobulin and albumin, comprising:

- contacting the blood-derived plasma sample with medium chain fatty acids under conditions of pH ranging from about 4.2 to about 5.0 to allow albumin to selectively precipitate from the blood-derived plasma sample,

- thereby forming a suspension comprising a soluble protein-containing component comprising immunoglobulins and an insoluble protein-containing component comprising albumin, and

- Separating the soluble protein-containing component from the insoluble protein-containing component to obtain a purified immunoglobulin solution and a suspension comprising albumin.

The step of separating the soluble protein-containing component from the insoluble protein-containing component preferably includes subjecting the suspension to sequential extractive filtration. Preferably, separating the soluble protein-containing component from the insoluble protein-containing component comprises:

- feeding the suspension to a filtration unit comprising a dynamic filter element configured to produce a first retentate and a first filtrate, and

- recovering the first filtrate and the first residue.

The first retentate comprising the insoluble protein-containing component is generally rich in albumin, and the first filtrate comprising the soluble protein-containing component is rich in immunoglobulins.

The first filtrate enriched in immunoglobulins may be subjected to a concentration step prior to further processing. The concentration step may include a continuous concentration process, whereby the first filtrate is fed to a second filtration unit, said second filtration unit being configured to filter a second immunoglobulin-enriched retentate and a second immunoglobulin-depleted retentate. and a cross-flow filter element or TFF configured to produce liquid. Optionally, a second filtrate depleted in immunoglobulins can be streamed back to the first tank and/or a second retentate enriched in immunoglobulins can be streamed back to the second tank.

As further described herein, it will be appreciated that solutions enriched in immunoglobulins can be further purified using standard techniques.

In a preferred embodiment, a method for purifying immunoglobulins and albumin comprising:

- To allow albumin to selectively precipitate from the blood-derived plasma sample, the blood-derived plasma sample and medium chain fatty acids are mixed under conditions where the pH range is from about 4.2 to about 5.0 and the conductivity is from about 8 to about 12 mS/cm. contacting step,

- thereby forming a suspension comprising a soluble protein-containing component comprising immunoglobulins and an insoluble protein-containing component comprising albumin, and

- separating the soluble protein-containing component from the insoluble protein-containing component to obtain a purified immunoglobulin solution,

- Optionally, the step of separating the soluble protein-containing component from the insoluble protein-containing component comprises feeding the suspension to a filtration unit comprising a dynamic filter element configured to produce a first retentate and a first filtrate, and 1 A method for purifying immunoglobulin and albumin is provided, comprising recovering the filtrate.

If the pH needs to be further adjusted, the pH of the blood-derived plasma sample can be adjusted without substantially diluting the sample, for example, with concentrated acid (e.g., acetic acid) or acid and base (e.g., NaOH). ) can be adjusted directly by adding a combination of Likewise, the conductivity of blood-derived plasma can be adjusted directly without substantially diluting the sample.

In a sixth aspect, a method for purifying immunoglobulins and albumin from plasma, comprising:

a) In a first tank, the blood-derived plasma sample is placed under conditions of a pH ranging from about 4.2 to about 5.0 and optionally a conductivity of from about 8 to about 12 mS/cm to allow selective precipitation of albumin from the blood-derived plasma sample. mixing the derived plasma sample and medium chain fatty acids to form a suspension comprising soluble immunoglobulins and insoluble albumin;

b) feeding the suspension to a first filtration unit, wherein the first filtration unit is configured to produce a first retentate comprising the insoluble albumin and a first filtrate comprising the soluble immunoglobulin. The supply step, including,

c) optionally streaming the first residue into the first tank to dilute the suspension in the first tank,

d) recovering the first filtrate from a second tank, and

A method is provided for purifying immunoglobulins and albumin from plasma, comprising the step of e) optionally concentrating said first filtrate.

Accordingly, in a seventh aspect, the present invention provides a method for obtaining an albumin precipitate, comprising:

- contacting the blood-derived plasma sample with medium chain fatty acids under conditions allowing albumin to selectively precipitate from the blood-derived plasma sample,

- the conditions are a pH range of about 4.15 to about 4.25,

- It provides for obtaining an albumin precipitate, whereby an albumin precipitate is formed.

Preferably, the conditions are also such that the conductivity is from about 5 to about 12 mS/cm, preferably from 8 to about 12 mS/cm. More preferably, when the blood-derived plasma is diluted, the conductivity is greater than 5 mS/cm and less than 12 mS/cm or less than about 12 mS/cm. When blood-derived plasma is not diluted, the conductivity is greater than or equal to 8 mS/cm but less than 12 mS/cm or less than about 12 mS/cm.

In an eighth aspect, the present invention provides a method for purifying albumin, comprising:

- contacting the blood-derived plasma sample with medium chain fatty acids under conditions of a pH ranging from about 4.15 to about 4.25 to allow albumin to selectively precipitate from the blood-derived plasma sample,

- thereby forming a suspension comprising a soluble protein-containing component comprising immunoglobulins and an insoluble protein-containing component comprising albumin, and

- Separating the soluble protein-containing component from the insoluble protein-containing component to obtain a precipitate or suspension containing albumin.

The step of separating the soluble protein-containing component from the insoluble protein-containing component preferably includes subjecting the suspension to sequential extractive filtration. Preferably, separating the soluble protein-containing component from the insoluble protein-containing component comprises:

- feeding the suspension to a filtration unit comprising a dynamic filter element configured to produce a first retentate and a first filtrate, and

- Comprising recovery of the first residue.

The first retentate comprising the insoluble protein-containing component is generally rich in albumin, and the first filtrate comprising the soluble protein-containing component is rich in immunoglobulins.

As further described herein, it will be appreciated that the albumin-rich first residue can be further purified using standard techniques.

In a preferred embodiment, a method for purifying albumin comprising:

- To allow albumin to selectively precipitate from the blood-derived plasma sample, the blood-derived plasma sample and medium chain fatty acids are subjected to a pH range of about 4.15 to about 4.25 and a conductivity of about 8 to about 12 mS/cm. contacting step,

- thereby forming a suspension comprising a soluble protein-containing component comprising immunoglobulins and an insoluble protein-containing component comprising albumin, and

- separating the soluble protein-containing component from the insoluble protein-containing component to obtain a suspension comprising albumin,

- Optionally, the step of separating the soluble protein-containing component from the insoluble protein-containing component comprises feeding the suspension to a filtration unit comprising a dynamic filter element configured to produce a first retentate and a first filtrate, and 1 A method for purifying albumin is provided, comprising recovering the filtrate.

If the pH needs to be further adjusted, the pH of the blood-derived plasma sample can be adjusted without substantially diluting the sample, for example, with concentrated acid (e.g., acetic acid) or acid and base (e.g., NaOH). ) can be adjusted directly by adding a combination of Likewise, the conductivity of blood-derived plasma can be adjusted directly without substantially diluting the sample.

In a ninth aspect, a method for purifying albumin from plasma, comprising:

a) In a first tank, the blood-derived plasma sample is placed under conditions of a pH ranging from about 4.15 to about 4.25, optionally with a conductivity of about 8 to about 12 mS/cm, to allow selective precipitation of albumin from the blood-derived plasma sample. mixing the derived plasma sample and medium chain fatty acids to form a suspension comprising soluble immunoglobulins and insoluble albumin;

b) feeding the suspension to a first filtration unit, wherein the first filtration unit is configured to produce a first retentate comprising the insoluble albumin and a first filtrate comprising the soluble immunoglobulin. The supply step, including,

c) optionally streaming the first residue into the first tank to dilute the suspension in the first tank,

d) recovering said first residue. A method is provided for purifying albumin from plasma.

In some embodiments, the residual suspension or first retentate of the first tank contains insoluble albumin complexed with medium chain fatty acids. In order to disrupt the binding between albumin and medium chain fatty acids, the pH of the residual suspension or first residue can be adjusted to 6.4 to 6.7, preferably 6.8 to 7.2. In one aspect, the pH can be adjusted with 1M sodium hydroxide. Optionally, the first retentate is subjected to mixing until the pH of the dissolved albumin stabilizes (generally about 30 to 60 minutes). The solution may then be fed to a further filtration unit (e.g. a second system comprising a further filtration unit, e.g. an additional filtration unit corresponding to 5 shown in Figure 16), The filtration unit of includes a dynamic filter element for producing an albumin-depleted retentate and an albumin-enriched filtrate.

In one embodiment, the albumin-enriched filtrate is preferably filtered using a TFF membrane (e.g., a second system comprising an additional filtration unit, e.g., 8 shown in Figure 16). Concentrated sequentially to up to 20 to 45 g/L protein or to about 20 to 45 g/L protein using , to form a concentrated albumin solution.

The concentrated albumin solution may then be heated in the range of 60 to 65° C. for a period of time typically greater than 90 minutes. Without wishing to be bound by any theory, it is believed that various proteins other than albumin are denatured in this step.

After heating the concentrated albumin solution, the pH of the solution can be generally adjusted to 4.20 or about 4.20 using 1M hydrochloric acid. At the same time, the concentrated albumin solution can be cooled to 4° C., thereby forming a precipitate of proteins denatured during the heating step. The precipitate may then be removed by filtration, whereby the filtrate contains purified albumin (generally albumin containing at least 95% total protein, at least 96% total protein, at least 97% total protein, or at least 98% total protein). purity and albumin yield of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, or at least 90%).

In certain embodiments of the invention, the dynamic filter element of the first filtration unit configured to produce a filtrate (permeate) rich in soluble proteins (immunoglobulins) is a dynamic cross-flow filter element.

In some embodiments of the invention, the dynamic filter element of the second filtration unit configured to produce an immunoglobulin-enriched retentate is a dynamic crossflow filter element.

In an optional embodiment of the invention, the dynamic filter element of the further filtration unit configured to produce albumin-enriched filtrate is a dynamic crossflow filter element.

In a preferred embodiment, the dynamic cross-flow filter element is a rotating cross-flow filter element. More preferably, the rotating cross filter element comprises a filter disk. The filter disk is generally mounted on a shaft member. In one aspect, the rotating cross filter element includes at least one filter disk and at least one shaft member.

According to a preferred embodiment of any aspect of the invention, the filter disk membrane is a ceramic membrane. More preferably, the ceramic membrane has a pore size in the range of 5 nm to 2 μm. In certain embodiments, the ceramic membrane has a pore size of about 0.2 to 2 μm. In certain embodiments, the ceramic filter membrane has an average pore size in the range of 5 nm to 200 nm (0.2 μm). In certain embodiments, the ceramic filter membrane has an average pore size of at least 50 nm and at most 100 nm. These filter discs are supplied by Kerafol and Flowserve.

It will be understood that a blood-derived plasma sample may include any plasma sample derived from blood, preferably human blood. In certain embodiments, the blood-derived plasma sample comprises fresh plasma, cryo-poor plasma, or cryo-rich plasma. Plasma may be obtained from multiple donors and/or subjects and may be pooled. The plasma may be hyperimmune plasma.

Preferably, the plasma sample does not include filter preparation and/or has not been subjected to an alcohol fractionation process or other fractionation process.

In any of the first, second or third aspects, the pH of the plasma sample is adjusted to a pH range of about 4.6 to about 5.0 prior to contacting and/or mixing with the medium chain fatty acids. For example, the pH of the plasma sample can be adjusted to a pH of about 4.6, about 4.7, about 4.8, about 4.9, about 5.0. The pH of the plasma sample can be adjusted to pH 4.6, pH 4.7, pH 4.8, pH 4.9 or pH 5.0.

In any of the fourth, fifth or sixth aspects, the pH of the plasma sample is adjusted to a pH range of about 4.2 to about 5.0 prior to contacting and/or mixing with the medium chain fatty acids. For example, the pH of the plasma sample can be adjusted to a pH of about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, or about 5.0. The pH of the plasma sample can be adjusted to pH 4.2, pH 4.3, pH 4.4, pH 4.5, pH 4.6, pH 4.7, pH 4.8, pH 4.9 or pH 5.0.

In any of the seventh, eighth or ninth aspects, the pH of the plasma sample is adjusted to a pH range of about 4.15 to about 4.25 prior to contacting and/or mixing with the medium chain fatty acids. For example, the pH of the plasma sample can be adjusted to a pH of about 4.15, about 4.16, about 4.17, about 4.18, about 4.19, about 4.20, about 4.21, about 4.22, about 4.23, about 4.24, or about 4.25. The pH of the plasma sample can be adjusted to pH 4.15, pH 4.16, pH 4.17, pH 4.18, pH 4.19, pH 4.20, pH 4.21, pH 4.22, pH 4.23, pH 4.24 or pH 4.25.

In some embodiments, the pH of a plasma sample can be adjusted without substantially diluting the plasma prior to contacting it with medium chain fatty acids. This pH adjustment can be accomplished by adding a concentrated acid, such as acetic acid, or a combination of an acid and a base (e.g., NaOH), if the pH needs to be further adjusted. If necessary, the conductivity of the resulting solution is adjusted to achieve a desired conductivity of about 8 to about 12 mS/cm.

In certain aspects of any aspect of the invention, the plasma sample may be diluted in a buffer prior to mixing with medium chain fatty acids. The dilution rate of plasma may be about 1:0.5, about 1:0.75, about 1:1, about 1:1.25, about 1:1.5, about 1:1.75 or about 1:2. As a result, the plasma may be diluted and have a conductivity of about 5 to about 12 mS/cm.

The buffer for diluting plasma may be any buffer suitable for dilution of plasma, such as acetate buffer (eg, sodium acetate). The acetate buffer may include sodium acetate trihydrate and glacial acetic acid. The concentration of the buffer can be 60mM, 80mM, 100mM or 0.22M and the pH is 4.1 or about 4.1, 4.2 or about 4.2, 4.3 or about 4.3, 4.4 or about 4.4, 4.5 or about 4.5, 4.6 or about 4.6, 4.7 or It may be about 4.7, 4.8 or about 4.8. Alternatively, phosphate-acetate buffer may be used. The phosphate-acetate buffer may include 10mM phosphate (eg, sodium phosphate) and 10mM acetate (eg, sodium acetate). The pH of this buffer may be about 4.3 to about 4.4. If the conductivity of the resulting solution needs to be adjusted to achieve a desired conductivity of about 5 to about 12 mS/cm, preferably about 8 to about 12 mS/cm, this may be achieved, for example, by using a more concentrated acetate buffer (e.g. , can be achieved by adding 3.5M acetate buffer containing sodium acetate trihydrate and glacial acetic acid, pH 5).

Typically, buffers are used to dilute plasma as well as facilitate pH adjustment of plasma samples. Accordingly, in any of the first, second or third aspects of the invention, the pH of the diluted plasma sample is from about 4.6 to about 5.0. For example, the pH of the diluted plasma sample may be a pH of about 4.6, about 4.7, about 4.8, about 4.9, about 5.0. The pH of the diluted plasma sample may be pH 4.6, pH 4.7, pH 4.8, pH 4.9, or pH 5.0. Accordingly, in any of the fourth, fifth or sixth aspects of the invention, the pH of the diluted plasma sample is from about 4.2 to about 5.0. For example, the pH of the diluted plasma sample may be a pH of about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, or about 5.0. The pH of the diluted plasma sample may be pH 4.2, pH 4.3, pH 4.4, pH 4.5, pH 4.6, pH 4.7, pH 4.8, pH 4.9, or pH 5.0. Accordingly, in any of the seventh, eighth or ninth aspects of the invention, the pH of the diluted plasma sample is from about 4.15 to about 4.25. For example, the pH of the diluted plasma sample may be a pH of about 4.15, about 4.16, about 4.17, about 4.18, about 4.19, about 4.20, about 4.21, about 4.22, about 4.23, about 4.24, or about 4.25. The pH of the diluted plasma sample may be pH 4.15, pH 4.16, pH 4.17, pH 4.18, pH 4.19, pH 4.20, pH 4.21, pH 4.22, pH 4.23, pH 4.24, or pH 4.25.

In any aspect or embodiment, the conditions may be a conductivity of from about 5 to about 12 mS/cm, preferably from 8 to about 12 mS/cm. More preferably, when the blood-derived plasma is diluted, the conductivity is greater than 5 mS/cm and less than 12 mS/cm, or less than about 12 mS/cm. When blood-derived plasma is undiluted, the conductivity is greater than or equal to 8 mS/cm but less than 12 mS/cm or less than about 12 mS/cm.

For example, the conductivity of a diluted or undiluted plasma sample is about 8 to about 12 mS/cm. Accordingly, in some embodiments, the conductivity of the diluted or undiluted plasma sample is about 8 mS/cm, about 9 mS/cm, about 10 mS/cm, about 11 mS/cm, or about 12 mS/cm. In some embodiments, the diluted or undiluted plasma sample has a conductivity of 8 mS/cm, 9 mS/cm, 10 mS/cm, 11 mS/cm, or 12 mS/cm. In some embodiments, the conductivity of the diluted plasma is at least 5 mS/cm, 6 mS/cm, or 7 mS/cm. Generally, conductivity is measured at room temperature, preferably between 18 and 25°C.

In some embodiments, the medium chain fatty acids may be selected from fatty acids with the formula CH ₃ (CH ₂ ) _n COOH, wherein the fatty acids are C4 to C10 carboxylic acids. Fatty acids can be saturated or unsaturated. More preferably, the fatty acid includes enanthic (heptanoic) acid, caprylic (octanoic) acid, octenoic acid, pelargonic (nonane) acid, nonenoic acid or capric (decanoic) acid. Most preferably the fatty acid is caprylic (octanoic) acid. Also contemplated as reagents are salts or esters of any of the fatty acids described herein, such as caprylate.

In any of the first, second and third aspects, the amount of fatty acid, preferably caprylic (octanoic) acid, mixed with the plasma sample is about 0.30 g/g total protein (in plasma sample), about 0.35 g/g total protein (in plasma sample). g/g total protein, about 0.40 g/g total protein, about 0.45 g/g total protein, or about 0.50 g/g total protein. Preferably, the amount of fatty acid, preferably caprylic (octanoic) acid, is about 0.300 g/g total protein, about 0.325 g/g total protein, about 0.350 g/g total protein, about 0.375 g/g total protein, about 0.400 g/g total protein, about 0.425 g/g total protein, or about 0.450 g/g total protein. More preferably, the amount of fatty acids, preferably caprylic (octanoic) acid, is at least about 0.350 g/g total protein. Preferably, the amount of fatty acids, preferably caprylic (octanoic) acid, ranges from about 0.38 to about 0.50 g/g total protein. More preferably, the amount of fatty acid, preferably caprylic (octanoic) acid, is about 0.38 g/g total protein, about 0.39 g/g total protein, about 0.40 g/g total protein, about 0.41 g/g total protein, About 0.42 g/g total protein, about 0.43 g/g total protein, about 0.44 g/g total protein, about 0.45 g/g total protein, about 0.46 g/g total protein, about 0.47 g/g total protein, about 0.48 g/g total protein, about 0.49 g/g total protein, or about 0.50 g/g total protein.

In any of the first, second and third aspects, the amount of fatty acid, preferably caprylic (octanoic) acid, is 0.30 g/g total protein (in plasma sample), 0.35 g/g total protein, 0.40 g/g total protein. g/g total protein, 0.45 g/g total protein, or 0.50 g/g total protein. Preferably, the amount of fatty acid, preferably caprylic (octanoic) acid, is 0.300 g/g total protein, 0.325 g/g total protein, 0.350 g/g total protein, 0.375 g/g total protein, 0.400 g/g total. Protein, 0.425 g/g total protein or 0.450 g/g total protein. More preferably, the amount of fatty acids, preferably caprylic (octanoic) acid, is at least 0.350 g/g total protein. Preferably, the amount of fatty acids, preferably caprylic (octanoic) acid, ranges from 0.38 to 0.50 g/g total protein. More preferably, the amount of fatty acid, preferably caprylic (octanoic) acid, is 0.38 g/g total protein, 0.39 g/g total protein, 0.40 g/g total protein, 0.41 g/g total protein, 0.42 g/g Total protein, 0.43 g/g Total protein, 0.44 g/g Total protein, 0.45 g/g Total protein, 0.46 g/g Total protein, 0.47 g/g Total protein, 0.48 g/g Total protein, 0.49 g/g Total Protein or 0.50 g/g total protein.

In any of the fourth, fifth and sixth aspects, the amount of fatty acid, preferably caprylic (octanoic) acid, mixed with the plasma sample is about 0.35 g/g total protein (in plasma sample), about 0.40 g/g total protein (in plasma sample). g/g total protein, about 0.45 g/g total protein, about 0.50 g/g total protein, or about 0.55 g/g total protein. Preferably, the amount of fatty acid, preferably caprylic (octanoic) acid, is about 0.35 g/g total protein, about 0.36 g/g total protein, 0.37 g/g total protein, 0.38 g/g total protein, about 0.39 g. /g total protein, approximately 0.40 g/g total protein, approximately 0.41 g/g total protein, approximately 0.42 g/g total protein, approximately 0.43 g/g total protein, approximately 0.44 g/g total protein, approximately 0.45 g/g Total protein, approximately 0.46 g/g total protein, approximately 0.47 g/g total protein, approximately 0.48 g/g total protein, approximately 0.49 g/g total protein, approximately 0.50 g/g total protein, approximately 0.51 g/g total protein , about 0.52 g/g total protein, about 0.53 g/g total protein, about 0.54 g/g total protein, or about 0.55 g/g total protein.

In any of the fourth, fifth and sixth aspects, the amount of fatty acid, preferably caprylic (octanoic) acid, mixed with the plasma sample is 0.36 g/g/g total protein, 0.37 g/g total protein, 0.38 g/g total protein, 0.39 g/g total protein, 0.40 g/g total protein, 0.41 g/g total protein, 0.42 g/g total protein, 0.43 g/g total protein, 0.44 g/g total protein, 0.45 g/g total protein, 0.46 g/g total protein, 0.47 g/g total protein, 0.48 g/g total protein, 0.49 g/g total protein, 0.50 g/g total protein, 0.51 g/g total protein, 0.52 g /g total protein, 0.53 g/g total protein, 0.54 g/g total protein, or 0.55 g/g total protein.

In any of the seventh, eighth or ninth aspects, the amount of fatty acid, preferably caprylic (octanoic) acid, mixed with the plasma sample is at least about 0.35 g/g total protein. Preferably, the amount of fatty acid, preferably caprylic (octanoic) acid, mixed with the plasma sample is greater than about 0.35 g/g total protein and less than or equal to about 1.1 g/g total protein. The amount of fatty acids, preferably caprylic (octanoic) acid, mixed with the plasma sample can be about 0.35 g/g total protein, about 0.36 g/g total protein, about 0.37 g/g total protein, about 0.38 g/g total protein, About 0.39 g/g total protein, about 0.40 g/g total protein, about 0.41 g/g total protein, about 0.42 g/g total protein, about 0.43 g/g total protein, about 0.44 g/g total protein, about 0.45 g/g total protein, approximately 0.46 g/g total protein, approximately 0.47 g/g total protein, approximately 0.48 g/g total protein, approximately 0.49 g/g total protein, approximately 0.50 g/g total protein, approximately 0.51 g/g g total protein, approximately 0.52 g/g total protein, approximately 0.53 g/g total protein, approximately 0.54 g/g total protein, approximately 0.55 g/g total protein, approximately 0.56 g/g total protein, approximately 0.57 g/g total Protein, about 0.58 g/g total protein, about 0.59 g/g total protein, about 0.60 g/g total protein, about 0.61 g/g total protein, about 0.62 g/g total protein, about 0.63 g/g total protein, About 0.64 g/g total protein, about 0.65 g/g total protein, about 0.66 g/g total protein, about 0.67 g/g total protein, about 0.68 g/g total protein, about 0.69 g/g total protein, about 0.70 g/g total protein, approximately 0.71 g/g total protein, approximately 0.72 g/g total protein, approximately 0.73 g/g total protein, approximately 0.74 g/g total protein, approximately 0.75 g/g total protein, approximately 0.76 g/g g total protein, approximately 0.77 g/g total protein, approximately 0.78 g/g total protein, approximately 0.79 g/g total protein, approximately 0.80 g/g total protein, approximately 0.81 g/g total protein, approximately 0.82 g/g total Protein, about 0.83 g/g total protein, about 0.84 g/g total protein, about 0.85 g/g total protein, about 0.86 g/g total protein, about 0.87 g/g total protein, about 0.88 g/g total protein, About 0.89 g/g total protein, about 0.90 g/g total protein, about 0.91 g/g total protein, about 0.92 g/g total protein, about 0.93 g/g total protein, about 0.94 g/g total protein, about 0.95 g/g total protein, approximately 0.96 g/g total protein, approximately 0.97 g/g total protein, approximately 0.98 g/g total protein, approximately 0.99 g/g total protein, approximately 1.0 g/g total protein, approximately 1.01 g/g g total protein, approximately 1.02 g/g total protein, approximately 1.03 g/g total protein, approximately 1.04 g/g total protein, approximately 1.05 g/g total protein, approximately 1.06 g/g total protein, approximately 1.07 g/g total Protein, about 1.08 g/g total protein, about 1.09 g/g total protein, or about 1.1 g/g total protein.

In any of the fourth, fifth and sixth aspects, the amount of fatty acid, preferably caprylic (octanoic) acid, mixed with the plasma sample is 0.35 g/g total protein, 0.36 g/g total protein, 0.37 g/g total protein, 0.38 g/g total protein, 0.39 g/g total protein, 0.40 g/g total protein, 0.41 g/g total protein, 0.42 g/g total protein, 0.43 g/g total protein, 0.44 g /g total protein, 0.45 g/g total protein, 0.46 g/g total protein, 0.47 g/g total protein, 0.48 g/g total protein, 0.49 g/g total protein, 0.50 g/g total protein, 0.51 g/ g total protein, 0.52 g/g total protein, 0.53 g/g total protein, 0.54 g/g total protein, 0.55 g/g total protein, 0.56 g/g total protein, 0.57 g/g total protein, 0.58 g/g Total protein, 0.59 g/g total protein, 0.60 g/g total protein, 0.61 g/g total protein, 0.62 g/g total protein, 0.63 g/g total protein, 0.64 g/g total protein, 0.65 g/g total Protein, 0.66 g/g total protein, 0.67 g/g total protein, 0.68 g/g total protein, 0.69 g/g total protein, 0.70 g/g total protein, 0.71 g/g total protein, 0.72 g/g total protein , 0.73 g/g total protein, 0.74 g/g total protein, 0.75 g/g total protein, 0.76 g/g total protein, 0.77 g/g total protein, 0.78 g/g total protein. 0.79 g/g total protein, 0.80 g/g total protein, 0.81 g/g total protein, 0.82 g/g total protein, 0.83 g/g total protein, 0.84 g/g total protein, 0.85 g/g total protein, 0.86 g/g total protein, 0.87 g/g total protein, 0.88 g/g total protein, 0.89 g/g total protein, 0.90 g/g total protein, 0.91 g/g total protein, 0.92 g/g total protein, 0.93 g /g Total Protein, 0.94 g/g Total Protein, 0.95 g/g Total Protein, 0.96 g/g Total Protein, 0.97 g/g Total Protein, 0.98 g/g Total Protein, 0.99 g/g Total Protein, 1.0 g/ g total protein, 1.01 g/g total protein, 1.02 g/g total protein, 1.03 g/g total protein, 1.04 g/g total protein, 1.05 g/g total protein, 1.06 g/g total protein, 1.07 g/g Total protein, 1.08 g/g total protein, 1.09 g/g total protein, or 1.1 g/g total protein.

In a preferred embodiment, contacting the plasma sample with the medium chain fatty acid comprises mixing the plasma sample and the fatty acid to obtain a homogeneous emulsion of the medium chain fatty acid and the plasma sample. In a preferred embodiment, the mixing is vigorous to allow the formation of a homogeneous emulsion.

Preferably, the plasma sample and the medium chain fatty acids, preferably caprylic (octanoic) acid, are incubated for at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least Mix for a period of about 40 minutes, at least about 45 minutes, or at least about 50 minutes or longer.

Preferably, the mixing step is followed by an incubation period prior to the step of separating the soluble protein-containing component (soluble immunoglobulin) from the insoluble protein-containing component (insoluble albumin). The incubation period is preferably at least about 20 minutes, at least about 30 minutes, at least about 40 minutes, at least about 50 minutes, at least about 60 minutes, at least about 70 minutes, at least about 80 minutes, at least about 90 minutes, at least about 100 minutes. , at least about 110 minutes, at least about 120 minutes, at least about 130 minutes, at least about 140 minutes, at least about 150 minutes or more.

In any of the aspects of the invention, unless otherwise specified, the steps of the method are performed at a temperature of from about 18°C to about 37°C, preferably from about 18°C to about 24°C. In some embodiments, the temperature is about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, or about 24°C. In some embodiments, the temperature is 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, or 24°C.

The immunoglobulin purified according to any aspect of the invention preferably comprises immunoglobulin G (IgG), preferably human immunoglobulin G (IgG). The immunoglobulin may comprise any of the IgG subclasses IgG1, IgG2, IgG3 or IgG4, preferably the relative distribution of the IgG subclasses is similar to or substantially the same as the distribution of IgG subclasses normally observed in plasma. . Optionally, IgG1 is present in the composition in an amount of about 60 to about 70% of the total immunoglobulins, IgG2 is present in an amount of about 25 to about 35% of the total immunoglobulins, and IgG3 is present in an amount of about 2 to about 3% of the total immunoglobulins. , and IgG4 exists at about 0.5 to about 1.5% of the total immunoglobulin.

In any of the aspects of the invention, the immunoglobulin solution or concentrated immunoglobulin is further processed to further purify the immunoglobulin. Preferably, further processing does not involve the additional step of continuous filter extraction.

In certain embodiments, immunoglobulins are subjected to further processing, such as low pH treatment, chromatography steps (including anion exchange chromatography and/or immunoaffinity chromatography), virus filtration and inactivation steps, concentration and formulation, to obtain a final The product is administered, for example, to the human body. The final product can be used in the treatment of immune diseases, certain autoimmune diseases, and certain neurological diseases. These conditions include rheumatoid arthritis, systemic lupus erythematosus (SLE), antiphospholipid syndrome, immune thrombocytopenia (ITP), Kawasaki disease, Guillain-Barré syndrome (GBS), multiple sclerosis (MS), and chronic inflammatory demyelinating polyneuropathy (CIDP). ), multifocal motor neuropathy (MMN), myasthenia gravis (MG), skin bullous disease, scleroderma, dermatomyositis, polymyositis, Alzheimer's disease, Parkinson's disease, Alzheimer's disease associated with Down syndrome, cerebral amyloid angiopathy, Lewy body dementia , frontotemporal lobe degeneration, or vascular dementia. Additionally, the final IVIg and SCIg products may also be used in other medical procedures such as cell and organ transplantation.

In any of the embodiments of the invention, the purified immunoglobulin solution contains one or more of the following impurities: IgA, IgM, albumin, α(alpha)-2 macroglobulin, α(alpha)-1 antibody. Trypsin, lipids, lipoproteins.

In some embodiments, the insoluble protein-containing component of the suspension (i.e., obtained by contacting a plasma sample with a medium chain fatty acid) is retained after the step of separating the soluble protein-containing component from the insoluble protein-containing component.

It will be appreciated that insoluble protein-containing components may be used for the purpose of obtaining purified fractions of albumin, for example as described herein.

For example, in relation to the third, sixth or ninth aspect of the invention, if the suspension obtained from the mixture of plasma and medium chain fatty acids is fed through the first filtration unit, any residual suspension and/or the first The residue can be further processed to obtain purified albumin.

Accordingly, in any aspect of the invention, the method further comprises the following steps:

a) adjusting the pH of the insoluble protein-containing component comprising albumin to a pH of about 6.4 to about 7.2 (preferably about 6.8 to about 7.2) to obtain solubilized albumin,

b) optionally subjecting the solubilized albumin to further processing steps to remove impurities from the albumin, and

c) recovering purified albumin from solubilized albumin.

In relation to the third, sixth and ninth aspects of the invention, the insoluble albumin may be residual insoluble protein remaining in the first tank and/or may further comprise a first residue.

Adjusting the pH of the insoluble protein-containing component can be performed directly without substantially diluting the insoluble protein-containing component. This pH adjustment can be accomplished by adding a concentrated acid, such as acetic acid, or a combination of an acid and a base (e.g., NaOH), if the pH needs to be further adjusted. For example, the insoluble protein-containing component (e.g., the residual suspension in the first tank) is adjusted to a pH of 6.4 to 7.2 (preferably 6.8 to 7.2) using 1M sodium hydroxide or phosphate buffer (pH 7.1 to 7.4). It can be adjusted with . Additionally, a combination of 0.12M sodium hydroxide and phosphate buffer is also considered. If necessary, the conductivity of the resulting solution is adjusted to achieve a desired conductivity of about 8 to about 15 mS/cm.

Optionally, the step of adjusting the pH of the insoluble protein-containing component includes first contacting the insoluble protein-containing component (insoluble albumin) with a buffer solution having a pH of 7.1 to 7.4 and optionally a conductivity of about 8 to about 15 mS/cm to further Solubilized albumin by forming a suspension and adjusting the pH of the additional suspension to a pH of at least about 6.4, preferably neutral, more preferably about 6.4 to about 7.2, about 6.4 to about 6.7, or about 6.8 to about 7.2. Includes obtaining.

Any buffer capable of dissolving albumin by disrupting the binding between albumin and medium chain fatty acids, thereby liberating the fatty acids from albumin, is suitable for use in this step. For example, the buffer solution may have a pH of about 7 and a conductivity of about 8 to about 15 mS/cm. This step involves adding a buffer to the tank containing the suspension and adjusting the pH to about 7, preferably about 7.2 (especially at least about 6.4, more preferably about 6.4 to about 6.7, about 6.4 to about 7.2, most preferably This can be done by stirring until the temperature changes to about 6.8 to about 7.2 (e.g., for 5 minutes). An example of a suitable buffer is phosphate buffer. The phosphate buffer may include NaH ₂ PO ₄ .2H ₂ O and NaH ₂ PO ₄ .12H ₂ O. The concentration of the buffer solution may be 0.12M and the pH may be 7.3±0.2. Alternatively, pH adjustment can be performed directly using a base, such as NaOH, as described above.

Solubilized albumin can be subjected to further processing steps to remove impurities.

In one aspect of any aspect of the invention, further processing of the solubilized albumin comprises

- feeding the solubilized albumin to a filtration unit, wherein the filtration unit comprises a dynamic filter element configured to produce an albumin-depleted retentate and an albumin-enriched filtrate, and

- It includes the step of recovering the albumin-rich filtrate.

The albumin-enriched filtrate may optionally be subjected to a concentration step prior to further processing. The concentration step may include a continuous concentration process, whereby the filtrate is fed into a second filtration unit, the second filtration unit being a cross-flow filter configured to produce an albumin-rich retentate and an albumin-depleted filtrate. contains elements.

According to this aspect of the invention, the method for further processing the solubilized albumin comprises

d) providing solubilized albumin to the first tank,

e) feeding the solubilized albumin solution to a first filtration unit, wherein the first filtration unit comprises a dynamic filter element configured to produce an albumin-depleted retentate and a filtrate enriched in soluble albumin. ,

f) optionally streaming the residue into the first tank to dilute the solution in the first tank,

g) recovering the albumin-rich filtrate in a second tank, and

h) optionally concentrating the filtrate.

In any embodiment, step h) of concentrating the filtrate comprises subjecting the filtrate to a continuous concentration process in a second filtration unit, wherein the second filtration unit comprises an albumin-enriched retentate and an albumin-depleted filtrate. Contains a dynamic filter element or TFF configured to generate a Optionally, the albumin-depleted filtrate can be streamed back to the first tank and/or the albumin-rich retentate can be streamed back to the second tank.

Preferably, the dynamic filter element of the first filtration unit configured to produce an albumin-rich filtrate (permeate) is a dynamic crossflow filter element. Preferably, the dynamic filter element of the second filtration unit configured to concentrate the albumin solution to produce an albumin-enriched retentate is a dynamic cross-flow filter element or TFF.

In another embodiment, to further purify the albumin, the solubilized albumin fraction can be subjected to alcohol precipitation methods and/or chromatography as are generally known in the art. Depending on the nature of the contaminants present in the albumin, various purification schemes can be adopted. For example, albumin can be subjected to well-known fractionation processes, such as ethanol fractionation, to produce supernatant I, supernatant II+III, supernatant-IV-1, supernatant-IV-4, or fraction V. Albumin can be further pH adjusted, ultrafiltered/diafiltered, and pasteurized, according to the AlbuRx production process. Other methods of producing purified albumin are well known, as is done in the Albumex production process, where albumin is subjected to ion exchange chromatography, followed by gel filtration chromatography and pasteurization. Suitable albumin purification processes are described in Matejtschuk, P. et al (2000) British Journal of Anaesthesia 85(6); 887-95, and in the Australian Public Assessment Report for Albumin (human) (2017) Therapeutic Goods Administration, pages 8-9 (available online at: https://www.tga.gov.au/sites/default/files/ auspar-albumin-human-170502.pdf)].

As used herein, except where the context otherwise requires, the term “comprise” and variations of the term, e.g., “comprising,” “comprises,” and “comprised” are not intended to exclude additional additives, ingredients, integers or steps.

Further aspects of the invention and of those described in the preceding paragraphs will become apparent from the following description, given by way of example, and with reference to the accompanying drawings.

The following drawings are not necessarily drawn to scale, but instead focus generally on illustrating the principles of the various aspects. In the following description, various aspects of the invention are explained with reference to the following drawings.
Figure 1: Distribution of IgG subclasses in blood-derived plasma and in clarified filtrate containing purified immunoglobulins obtained according to the method of the invention.
Figure 2: Protease composition of blood-derived plasma; diluted pooled plasma; Incubation with octanoic acid at 0 h, 1 h, 2 h, and 3 h (T0, T1, T2, and T3, respectively); and a UF/DF filtrate containing purified immunoglobulin prepared according to the method of the present invention. Protease levels are expressed as nkat/L.
Figure 3: Impurities detected in plasma and clarified filtrate containing purified immunoglobulins obtained according to the method of the present invention.
Figure 4: Prekallikrein activator (PKA), factor IX (FIX) and factor )) of activity (IU/mL).
Figure 5: Concentration of albumin (g/L) in blood-derived plasma and clarified filtrate containing purified immunoglobulins obtained according to the method of the invention.
Figure 6: Protein composition of plasma and clarified filtrate containing purified immunoglobulins obtained according to the method of the invention. The relative percentages of gamma globulin, alpha/beta globulin and albumin are indicated.
Figure 7: Yield of IgG (g/L plasma) at different octanoic acid amounts and different pH at constant ionic strength.
Figure 8: Yield of albumin (g/L plasma) at different octanoic acid amounts and different pH at constant ionic strength.
Figure 9: Yield (g/L plasma) of IgG, albumin, IgA and IgM at different ionic strengths at constant pH.
Figure 10: Yield of IgG (g/L plasma) of clarified and concentrated octanoic acid filtrate at various pH and various ionic strengths.
Figure 11: Albumin yield (g/L plasma) of clarified and concentrated octanoic acid filtrate at various pH and various ionic strengths.
Figure 12: IgA yield (g/L plasma) of clarified and concentrated octanoic acid filtrate at various pH and various ionic strengths.
Figure 13: IgM yield (g/L plasma) of clarified and concentrated octanoic acid filtrate at various pH and various ionic strengths.
Figure 14: Product-related impurities (g/L plasma).
Figure 15: Impurity profile of heat-treated albumin (g/L plasma).
Figure 16: Schematic flow diagram overview of continuous extraction filtration system.
Figure 16 illustrates a schematic flow diagram overview of a system 100 and method according to one preferred aspect of the present invention. Plasma is placed in tank (1) and pH and conductivity are adjusted by adding buffer/acid, etc. OA is then added, and the OA suspension is incubated in tank (1). The OA suspension can be supplied to the first filtration unit (5) via a pump (2) of different types of pumps (e.g. piston pumps, rotary pumps, centrifugal pumps and membrane pumps) and flow rates in the pipe (12). A control valve (3) may be used. The first filtration unit 5 is equipped with a rotating hollow shaft on which a filter disk is mounted (the filtrate flows from outside the hollow shaft to the inside). The first filtration unit 5 is further set up with a height-adjustable scraper to keep the filter cake thickness constant to achieve a constant filtrate flow. The desired filtration pressure is controlled and adjusted by the overflow valve (unfiltered suspension outlet). Filter discs used can be ceramic membranes, deep filter layers and sintered porous metal filter discs. Once the vessel of the first filtration unit 5 has been filled with suspension, continuous pressure extraction and separation can begin. The first filtration unit 5, which may comprise a pressure unit/vessel, is provided with suitable internal settings and conditions to simultaneously increase the extraction efficiency and the filtration process. Extraction efficiency is increased through turbulent mixing in unit 5 without the use of a mixer. Nevertheless, it can be envisaged that additional mixers may be provided to help the extraction process by creating turbulence. Additionally, higher final dilution factors, e.g., 1:≥30 as disclosed herein, also increase extraction efficiency, resulting in higher protein (e.g., IgG) yield. Of course, other higher final dilution factors (above 70) are also conceivable.
The first filtrate flows through a flow meter 6 installed in the pipe (or channel) 14 and is collected in the second tank 7. The unfiltered suspension (eg first retentate) flows back through a regulated outlet (3) installed in the pipe (13) of the tank (1). Once the limited capacity of the second tank 7 has been reached, the UF concentration process 8 can be started in the second filtration unit. The first filtrate from the second tank 7 flows through pipe 15 to an ultrafiltration (UF) system 8 (e.g., tangential flow filtration (TFF) using a TFF membrane). The transmembrane pressure is set such that the permeate flow rate 17 is equal to or approximately equal to the first filtrate flow rate in pipe 14. The permeate (or second filtrate) of the UF system 8 flows back to the first tank 1 through the pipe (or line or channel) 17, while the retentate of the UF system 8 (or The second retentate (= concentrated protein) flows back to the second tank 7 via pipe 16.
According to the invention, the first process unit 5 is provided with one or more rotating filter discs, said rotating filter discs being used to produce a first retentate and a first permeate/filtrate, the first process unit (5) 5) and one or more first filter elements for turbulent mixing of the contents. The first retentate can be fed back to the first tank (1) via the channel (13) via the control valve (3), while the first permeate/filtrate can be fed back to the second tank (14) via another channel (14). It can be supplied as (7). The first filtration element may be a filtration membrane based on ceramic material and with a pore diameter of about 5 to 5,000 nm, preferably 20 to 100 nm, more preferably 30 to 80 nm. Additionally, it can be expected that inorganic membranes or any other suitable membranes may provide similar effects as ceramic based membranes. The first filtration unit 5 may be provided with a pressure control device 4, for example a pressure gauge, to regulate the internal pressure. Similarly, a flow meter 6 may be installed in the system of the present invention to measure the flow rate of the suspension or solution.

Reference will now be made in detail to specific embodiments of the invention. Although the invention has been described in conjunction with embodiments, it will be understood that the invention is not intended to be limited to these embodiments. On the contrary, the present invention is intended to cover all alternatives, modifications and equivalents that may fall within the scope of the invention as defined by the claims.

Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The invention is in no way limited to the methods and materials described. It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual characteristic features mentioned or apparent from the text or drawings. All of these different combinations constitute various alternative embodiments of the invention.

For the purposes of interpreting this specification, terms used in the singular also include the plural and vice versa.

The present invention relates to systems and methods for efficient purification of immunoglobulins, preferably IgG, from plasma. The invention advantageously uses plasma as starting material and allows the extraction of immunoglobulins from plasma in a single step without the need for alcohol-based precipitation methods.

A further advantage of the present invention can be the simultaneous purification of albumin from a starting plasma sample, providing a method to rapidly obtain substantially pure preparations of both immunoglobulins and albumin from plasma through a single precipitation and filtration step.

A particular advantage of the inventive method approach is the absence of any filtration aids, which advantageously ensures that protease activity is reduced early in the process, ensuring maximum protein recovery and yield. In part, the additional advantage of separating the immunoglobulin-containing and albumin-containing components of plasma arises by applying a single continuous extraction filtration method. Continuous extraction methods facilitate downstream processing using conditions that minimize loss of proteins of interest, for example, by reducing the total amount of reagents needed to precipitate impurities or proteins of interest.

Justice

The term “soluble protein-containing component” is intended to refer to the soluble or aqueous phase component that results after mixing a blood-derived plasma sample with medium chain fatty acids according to the method of the invention. Typically, the soluble protein-containing component is very rich in immunoglobulins and other proteins that remain soluble even after mixing of plasma and fatty acids.

The term “insoluble protein-containing component” is intended to refer to the water-insoluble component or solid phase component resulting after mixing medium chain fatty acids with a blood-derived plasma sample according to the method of the present invention. Generally, the insoluble protein-containing component includes precipitated proteins, primarily albumin, but also other contaminating proteins that are denatured after mixing of plasma and fatty acids.

The method of the present invention allows selective precipitation of albumin from blood-derived plasma samples. In a preferred embodiment, precipitation causes at least 50% of the albumin in the sample to precipitate. More preferably, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85% or at least About 90% or more settles.

“High yield” means that the yield of the protein of interest, e.g., immunoglobulin G or albumin (and other proteins and immunoglobulins), is at least 80%, at least 85%, or at least the amount of protein in the soluble or insoluble protein-containing component. means 95%, preferably at least 96%, more preferably at least 98%, and most preferably greater than 98%.

The concentration of immunoglobulins and/or albumin in a sample can be measured by any means known to those skilled in the art. It should be understood that the method used to measure immunoglobulin or albumin may vary depending on the nature of the sample. For example, if the sample is an albumin-containing precipitate, it will be appreciated that it may be necessary to dissolve the precipitate (or a sample thereof) in a suitable buffer prior to measurement. Examples of suitable assays for measuring proteins of interest include high pressure liquid chromatography (HPLC, e.g., size exclusion HPLC), enzyme-linked immunosorbent assay (ELISA), and quantitative immunoturbidimetry.

The pH and/or conductivity of any sample can be measured by methods known in the art. Generally, pH and/or conductivity are measured at room temperature, preferably pH and/or conductivity are measured at 18 to 25°C. The unit of conductivity measurement is mS/cm, and it can be measured using a standard conductivity meter. The conductivity of a solution can be changed by changing the ion concentration in the solution. For example, the concentration of buffering agent and/or salt (e.g., NaCl or KCl) in the solution can be varied to achieve the desired conductivity. Preferably, the salt concentration is modified to achieve the desired conductivity as described in the Examples below or elsewhere herein.

“About” or “approximately” in relation to a numerical value given in terms of percentage, pH, amount or time period or other reference means to include the numerical value within 10% of the specified value.

Large-scale or industrial-scale processes or systems

The large-scale or industrial scale relevant to the invention refers to a production procedure based on at least 200 L, preferably at least 500 L and even more preferably at least 2,000 L of starting material, for example human plasma. For example, a typical commercial plasma donor pool size used for industrial scale protein manufacturing is 2,500 to 6,000 L of plasma per batch. In certain embodiments of the invention, the precipitate is obtained from 2,500 to 6,000 L of plasma. Some commercial manufacturing processes may use much larger plasma donor pool sizes, including up to 7,500 L, up to 10,000 L, and/or up to 15,000 L of plasma.

The methods and systems of the present invention can be used for large-scale industrial scale applications as well as stand-alone systems and/or methods for small production scale applications (starting materials may be less than 200 L).

starting material

The method of the invention advantageously enables the use of blood-derived plasma as starting material for extracting immunoglobulins and albumin. The plasma may be fresh plasma, “normal” plasma, “hyperimmune” plasma, cryo-deficient plasma (also referred to as frozen supernatant), or cryo-enriched plasma. Optionally, the plasma is treated to remove components such as C1 inhibitor, PCC (prothrombin complex remnants) and/or AT-III. Plasma may be obtained from multiple donors and/or individuals and pooled.

The term “frozen supernatant” (also called cryoprecipitate-depleted plasma, cryoprecipitate-depleted plasma, etc.) refers to plasma from which cryoprecipitate has been removed (from whole blood donation or plasmapheresis). Cryoprecipitation is the first step in most plasma protein fractionation methods used today for large-scale production of plasma protein therapeutics. The method generally involves pooling thawed frozen plasma under controlled conditions (e.g., below 6° C.), and the precipitate is collected by filtration or centrifugation. A supernatant fraction, known to those skilled in the art as “frozen supernatant”, is generally retained for use. The resulting cryo-deficient plasma had reduced levels of factor VIII (FVIII), von Willebrand factor (VWF), factor XIII (FXIII), fibronectin, and fibrinogen. The frozen supernatant contains alpha 1-antitrypsin (AAT), apolipoprotein A-I (APO), antithrombin III (ATIII), prothrombin complex containing coagulation factors (II, VII, IX and X), albumin (ALB) and immunoglobulins. , provides common feedstock used to manufacture a variety of therapeutic proteins, including, for example, immunoglobulin G (IgG).

The term “cryo-rich plasma” refers to plasma (from a whole blood donation or plasmapheresis) that has been frozen and then thawed but without the cryoprecipitate removed.

If plasma is frozen for transport from the collection site, thaw the frozen plasma before centrifugation and collect it in a pooling tank. Freeze precipitate is removed through continuous centrifugation. Frozen-depleted plasma can be pumped into a stainless steel fractionation tank and sampled for in-process control.

Plasma may be hyperimmune plasma, whether pooled from more than one or hundreds of individuals or obtained from a single individual. For example, plasma can be obtained from the blood of an individual(s) who has developed an immune response to an infection or has recovered (and is therefore an otherwise healthy individual).

Dynamic Filter Element

In any aspect of the invention, the dynamic filter element filtration unit configured to separate soluble immunoglobulins from insoluble albumin is a dynamic cross-flow filter element. In a preferred embodiment, the dynamic cross-flow filter element is a rotating cross-flow filter element. More preferably, the rotating cross filter element comprises a filter disk. The filter disk is generally mounted on a shaft member. In one aspect, the rotating cross filter element includes at least one filter disk and at least one shaft member.

According to a preferred embodiment of any aspect of the invention, the filter disk membrane is a ceramic membrane. More preferably, the ceramic membrane has a pore size in the range of 5 nm to 2 μm. In certain embodiments, the ceramic membrane has a pore size of about 0.2 to 2 μm. In certain embodiments, the ceramic filter membrane has an average pore size in the range of 5 nm to 200 nm (0.2 μm). In certain embodiments, the ceramic filter membrane has an average pore size in the range of 50 nm to 100 nm. These filter discs are supplied by Kerafol and Flowserve.

It will be appreciated that a dynamic filter element filtration unit configured to separate soluble immunoglobulins from insoluble albumin may include a plurality of filter disk membranes. Accordingly, the method contemplates the use of 1, 2, 3, 4, 5, 6 or more filter disc membranes to separate soluble immunoglobulins from insoluble albumin. The plurality of filter disk membranes may have the same or different pore sizes.

In a preferred embodiment, the filtration unit comprises a pressure vessel. The suspension from the first tank can be continuously fed into the pressure vessel through the inlet port. A distribution manifold allows uniform distribution of the suspension into the container. Accordingly, in certain aspects, the pressure vessel includes a distribution manifold. In some aspects, the first filtration unit includes a rotating crosshair filter element. Preferably, the filter element comprises one or more filter disks evenly spaced along at least one hollow central collection shaft. Filter disks can be arranged horizontally or vertically. When oriented horizontally, the filter disks are spaced along a vertically oriented hollow collection shaft. The collection shaft and disk can rotate. The suspension in the pressure vessel may permeate the outer membrane of the rotating filter disk and be channeled through the hollow central portion of the disk to a central collection shaft. Typically, filtrate (eg, containing partially purified immunoglobulins) may be removed from the shaft portion of the first filtration unit through a flanged port. Residue (including insoluble components) retained in the pressure housing can be fed out of the vessel through the outlet port. Typically, the residue is recycled to the first tank to dilute the suspension. In this way, the residue from the first filtration unit can be used to dilute the suspension in the first tank.

Dynamic cross-flow filtration, such as rotary filtration, provides maximum filtration efficiency. The cross-flow effect (tangential flow purification of the filter surface) is created by rotating the filter disk, rather than pumping a large volume across a fixed membrane as used in conventional (static) cross-flow filtration systems. The extreme cross-flow velocities generated on the surface of the rotating filter disk ensure highly efficient purification of the filter surface, while consuming a very small amount of energy compared to conventional cross-flow technologies.

Dynamic filter elements may also be used to perform a continuous concentration process. These dynamic filter elements typically include one or more ultrafiltration or diafiltration membranes.

A crossflow filter element for performing a continuous concentration process may include a dynamic ultrafiltration filter unit. Alternatively, the process involves a static ultrafiltration device, such as tangential flow filtration (TFF).

In a preferred embodiment of the invention, the dynamic cross-flow filter element or ultrafiltration filter unit for carrying out the concentration process comprises a molecular weight of the protein of interest (e.g. immunoglobulin G in the case of step e of the third aspect of the invention). and membranes with smaller molecular weight cutoffs. The molecular cutoff may be at least 3-fold lower than the molecular weight of the protein of interest (e.g., for a protein with a molecular weight of about 150 kDa, the membrane may have a cutoff of about 30 to 50 kDa). In this embodiment, the membrane cutoff is selected to retain the protein of interest during the enrichment process. As a general guideline, choose a nominal membrane cutoff that is at least 3 times lower than the molecular weight of the protein of interest to ensure that the protein is retained in the residue.

In one alternative embodiment, the dynamic cross-flow filter element or static ultrafiltration filter element for performing the concentration process comprises a membrane whose molecular weight cutoff is greater than the molecular weight of the protein of interest. In this embodiment, a nominal membrane cutoff is selected to ensure that the protein of interest passes through the membrane and is collected in the filtrate rather than the retentate.

In embodiments where the cross-flow filter element is a dynamic cross-flow filter element, preferably the element is a rotating cross-flow filter element configured to carry out a continuous concentration process.

According to a more preferred embodiment, the filtration element for carrying out the continuous concentration process has an average pore size of 5 to 5,000 nm, preferably 5 to 2,000 nm, 5 to 1,000 nm, 5 to 500 nm, 5 to 200 nm, 7 to 1,000 nm. , more preferably 7 to 500 nm, even more preferably 7 to 100 nm, most preferably 7 to 80 nm. Of course, the average pore size may be any other combination of the ranges given above. Filter manufacturers often assign terms such as nominal or average pore size ratings to commercial filters, which generally indicate that they meet specific retention criteria for particles or microorganisms rather than the actual geometric size of the pores.

In one aspect, the filtration element that performs the continuous concentration process is dynamic flow filtration, or tangential flow filtration using a TFF membrane.

In certain embodiments, a rotating crosshair filter element for performing a continuous concentration process includes a filter disk (eg, a ceramic disk). In some embodiments, the filter disk includes a membrane having an average pore size of a microfilter. In another aspect, the filter disk includes a membrane having an average pore size of an ultrafilter. In a further aspect, the filter disk includes a membrane having an average pore size of a diafilter. In one aspect, the average pore size of the filter disk membrane ranges from 5 nm to 2 μm. In certain embodiments, the average pore size of the filter disk membrane ranges from 50 nm to 500 nm (i.e., 0.5 μm). In some embodiments, the filter disk membrane has an average pore size in the range of 50 nm to 100 nm, 60 nm to 90 nm, or 60 nm to 80 nm. In some embodiments, the filter disk membrane has an average pore size of 60 nm or 80 nm.

In a particularly preferred embodiment, a rotating crosshair filter element for carrying out a continuous concentration process comprises a plurality of ceramic disks with pore sizes suitable for ultrafiltration and/or diafiltration. For example, the element preferably comprises at least one ceramic membrane with a pore size of 3 nm. Alternatively, the element preferably comprises at least one ceramic membrane with a pore size of 5 nm. Alternatively, the element preferably comprises at least one ceramic membrane with a pore size of 7 nm. Alternatively, the element preferably comprises at least one ceramic membrane with a pore size of 30 nm. The element may include a plurality of ceramic disks with various pore sizes, including ceramic disks with pore sizes of 3 nm and 5 nm. The element may include a plurality of ceramic disks with various pore sizes, including ceramic disks with pore sizes of 5 nm and 7 nm. The element may include a plurality of ceramic disks with various pore sizes, including ceramic disks with pore sizes of 3 nm and 30 nm. The element may include a plurality of ceramic disks with various pore sizes, including ceramic disks with pore sizes of 3 nm, 5 nm, 7 nm, and 30 nm.

According to a further preferred embodiment, the filtration element for carrying out the continuous concentration process comprises an ultrafiltration unit comprising a membrane in the form of a polymeric membrane, for example polyethersulfone or regenerated cellulose.

Dynamic cross-flow filtration, such as rotary filtration, provides maximum filtration efficiency. The cross-flow effect (tangential flow purification of the filter surface) is generated by rotating the filter disk, rather than pumping a large volume across a fixed membrane as used in conventional (static) cross-flow filtration systems. The extreme cross-flow velocities generated on the surface of the rotating filter disk ensure highly efficient purification of the filter surface, while consuming a very small amount of energy compared to conventional cross-flow technologies.

In the dynamic cross-flow filtration units and systems of the present invention, the rotating ceramic filter disk is generally assembled in a pressurized housing. The design of the disc shows internal drainage channels. The filtrate is transported from the outside of the disc to the inside of the disc. The rotation of the disk generates a shear force on the membrane surface. Using these techniques, high filtration fluxes can be achieved by preventing the build-up of filter cake. Some of the key parameters of rotary filtration are the rotational speed at which the ceramic filter disk rotates, solids content (concentration of the liquid resulting from filtrate removal), and transmembrane pressure. The transmembrane pressure is generally 0.1 to 2.5 bar, preferably 0.2 to 2.4 bar, more preferably 0.4 to 2.0 bar, 0.5 to 1.8 bar, 0.6 to 1.6 bar, 0.6 to 1.5 bar, 0.7 to 1.5 bar, and most preferably Preferably it is 0.8 to 1.5 bar. According to another embodiment, a pressure of at most 2 bar, preferably 0.1 to 2.0 bar or about 1.5 bar, 1.0 bar or 0.5 bar is provided to the filter unit.

Temperature affects the viscosity of protein solutions and therefore the flux during filtration using membranes. The starting suspension used in the process of the invention preferably has a temperature in the range from 0° C. to below the temperature at which the protein in question is denatured. Temperatures generally range from about 18 to about 40° C. or less. In certain embodiments, the temperature ranges from about 18 degrees Celsius to about 35 degrees Celsius or less. According to one preferred embodiment, the temperature of the suspension tank (i.e., the tank containing the fatty acid, preferably caprylic (octanoic) acid, for mixing with the plasma sample) is from about 18 to about 40° C. More preferably, the fatty acids, preferably caprylic (octanoic) acid, are mixed with the plasma in a second suspension tank at a temperature of about 18°C to about 24°C, optionally about 21°C, about 22°C, or about 23°C.

According to a further preferred embodiment, the temperature of the filter unit is preferably controlled between 2 and 25° C., more preferably between about 18 and about 24° C. These temperatures ensure optimal extraction and separation processes while maintaining the bioreactivity of the protein of interest throughout the process.

Filtration is carried out at a transmembrane filtration pressure below that which the membrane can withstand, for example from about 0.2 to about 3 bar, depending on the membrane material used in the invention. The transmembrane pressure is generally 0.1 to 2.5 bar, preferably 0.2 to 2.4 bar, more preferably 0.4 to 2.0 bar, 0.5 to 1.8 bar, 0.6 to 1.6 bar, 0.6 to 1.5 bar, 0.7 to 1.5 bar, and most preferably Preferably it is 0.8 to 1.5 bar. According to another embodiment, a pressure of at most 2 bar, preferably 0.1 to 2.0 bar or about 1.5 bar, 1.0 bar or 0.5 bar is provided to the filter unit.

According to another aspect, the continuous extraction process in a filter unit configured to separate impurities/precipitates from the first and second suspensions comprises a flow rate and/or residence time and/or impurities/precipitates of the suspension or solution into the filter unit. This is further supported by adjusting the flow rate of the retentate/raffinate and/or the flow rate of the first permeate/filtrate enriched in the protein of interest. For example, in one aspect, the linear velocity of the suspension or solution into the pressure vessel (filtration process unit) may be about 0.27 to 1.66 m/s. In another example, the linear velocity of the residue containing impurities/precipitates may be 0.25 to 1.33 m/s. In another example, the linear velocity of the permeate/filtrate enriched in the protein of interest may be 0.03 to 0.33 m/s. Multiplying the linear velocity by the cross-sectional area gives the volumetric flow rate. Additionally, turbulence may be created in the first process unit due to the speed of the rotating filter disk, which speed (often expressed as tangential speed) may be about 1 to 7 m/s. According to aspects of the invention, the speed of the rotating disk filter is 1 to 10 m/s. In a preferred embodiment of the invention, the speed of the rotating disk filter is 5 to 7 m/s. More preferably, the speed of the rotating disk filter is 7 m/s at 60 Hz (800 rpm). The rotational speed of the rotating cross filter element is about 600 rpm (50 Hz) to about 1,600 rpm (100 Hz), preferably about 800 rpm (60 Hz) to about 1,200 rpm (80 Hz), preferably about 800 rpm (60 Hz), about 1,000 rpm ( 70 Hz) or approximately 1,200 rpm (80 Hz). The rotational speed (Hz) used herein is intended to represent the speed of the motor. This can be correlated to speed in rpm using an appropriate calibration curve.

Using the above method, a continuous extraction and separation process can be implemented to maximize recovery of the protein of interest from the starting precipitate/material (i.e., blood-derived plasma). Thanks to the extraction process, almost all proteins of interest are extracted and recovered in subsequent steps. The method allows the liquid or diluent, such as buffer or water, to be recycled in a closed system, so that the amount of liquid is maintained throughout the process while the footprint (i.e. large tank volume) is reduced.

In another aspect, the invention includes a backflushing step with dynamic crossflow filtration to flush out contaminants that may accumulate in the system. Generally, the methods and systems of the present invention involve alternating filtration and backflushing such that backflushing periods occur periodically during which filtration is temporarily stopped (i.e., the feed pump is stopped) while liquid flow into the filtration system is reversed. do.

The frequency, duration and flow rate of backflushing may be adjusted to maximize filtration efficiency, with the understanding that a period of filtration is required prior to backflushing.

In certain preferred embodiments, the frequency of backflushing (and consequently the filtration period) is determined based on the total protein concentration or number of impurities in the starting material. As a result, backflushing is more frequently required when filtering suspensions containing plasma and fatty acids compared to the subsequent steps of further purification of the solubilized albumin preparation (which has relatively fewer impurities) to be filtered. You will understand. That is, as the total protein concentration and turbidity of the filtrate decrease, the frequency of backflushing intervals also decreases (i.e., the period of time between backflushing increases, and filtration proceeds for a longer period of time before a backflushing is needed). possible).

Protein concentration and turbidity of the filtrate can be monitored by various methods known in the art. In certain embodiments, the methods and systems of the invention include the use of an in-line detection unit that allows measuring protein concentration and/or turbidity as the filtrate passes into and/or out of the filtration unit. In a further embodiment, a dual wavelength photometer is used to detect protein concentration (e.g., by detecting the absorbance of the solution at a wavelength suitable for detecting protein concentration, e.g., in the range of 260 to 280 nm, preferably about 280 nm. ) and the turbidity of the solution (e.g., the absorbance of the solution at a wavelength suitable for detecting light scattering due to the presence of particulate matter, e.g., in the range of 400 to 900 nm, preferably in the range of about 600 to about 880 nm. Simultaneous evaluation of (by detecting the absorbance at the wavelength) can be facilitated. Dual wavelength photometric devices for use with chromatography and filtration units are well known in the art.

In certain examples, the frequency of backflushing may be every 15 seconds, 30 seconds, 45 seconds, 60 seconds, 75 seconds, 90 seconds, 105 seconds, 120 seconds, 135 seconds, 150 seconds, 200 seconds, 230 seconds, 260 seconds, 300 seconds. Intervals of seconds, 330 seconds, 360 seconds, 400 seconds, 1,000 seconds, 2,000 seconds, 3,000 seconds, 4,000 seconds or more.

It should be understood that the duration of backflushing intervals may vary depending on the filtration area and number of disks requiring backflushing. In general, the larger the filtration area, the larger the volume of backflushing buffer required, and the duration of backflushing is determined by the flow rate during backflushing. A skilled artisan can determine the appropriate duration, frequency and flow rate for backflushing depending on the size and number of disks of the system used. In certain examples, the backflushing duration is about 5 seconds, about 10 seconds, about 15 seconds, about 30 seconds, about 45 seconds, about 60 seconds or longer.

It will be appreciated that for practical reasons (and to maximize filtration efficiency) the duration of the backflushing interval is generally shorter than the duration of the filtration interval. In certain embodiments, the duration of the backflushing interval is at least 1/4, 1/8, 1/10, 1/16 or less than the duration of the filtration interval.

It will be further understood that the flow rate used during backflushing may be the same or different from the flow rate used for filtration. In certain embodiments, the flow rate during dynamic filtration ranges from about 15 to 100 L/hour, preferably from about 20 to 50 L/hour (about 200 ml/min to about 1 L/min, preferably from about 300 L/hour to about 900 ml/min, more preferably about 300 to 600 ml/min). Preferably, the flow rate for backflushing is lower than the flow rate used for filtration, such that in certain embodiments, the backflushing flow rate is about 100 to about 400 times slower than the flow rate used for filtration.

In certain embodiments, backflushing is performed using the same buffer contained in the first or second suspension. Alternatively, backflushing can be performed using the permeate obtained during the concentration process (e.g., when using ultrafiltration combined with dynamic cross-flow filtration to concentrate the filtrate obtained from dynamic cross-flow filtration). .

Recovery and further processing of proteins

The concentration of protein(s) in a sample (e.g., in a supernatant or a subsequently purified preparation thereof) can be measured by any means known to those skilled in the art. Examples of suitable assays include high pressure liquid chromatography (HPLC, e.g., size exclusion HPLC), enzyme-linked immunosorbent assay (ELISA), nephelometric methods, and immunoturbidimetry. This technique can be used to assess the purity of a sample (e.g., to identify the presence of undesirable protein contaminants, including proteases). Additionally, gel electrophoresis, such as SDS-PAGE with staining and densitometry, can be used to assess the purity of the sample and detect the presence of contaminating proteins. Reducing agents, such as dithiothreitol, can be used with SDS-PAGE to cleave disulfide-linked polymers.

In some embodiments, the temperature at which the conductivity of the solution is measured may be from about 4 to about 37°C, and preferably the temperature is from about 18 to about 25°C or from about 20 to about 25°C (room temperature).

The ultrafiltered product containing immunoglobulins (i.e. after continuous extraction and subsequent concentration) is later subjected to further processing, e.g. chromatography steps, virus inactivation steps, concentration and formulation, so that the final product is e.g. For example, it can be administered to the human body. The final product can be used in the treatment of immune conditions, certain autoimmune diseases, and certain neurological diseases. These conditions include rheumatoid arthritis, systemic lupus erythematosus (SLE), antiphospholipid syndrome, immune thrombocytopenia (ITP), Kawasaki disease, Guillain-Barré syndrome (GBS), multiple sclerosis (MS), and chronic inflammatory demyelinating polyneuropathy (CIDP). ), multifocal motor neuropathy (MMN), myasthenia gravis (MG), skin bullous disease, scleroderma, dermatomyositis, polymyositis, Alzheimer's disease, Parkinson's disease, Alzheimer's disease associated with Down syndrome, cerebral amyloid angiopathy, Lewy body dementia , frontotemporal lobe degeneration, or vascular dementia. Additionally, the final IVIg and SCIg products can also be used in other medical procedures, such as cell and organ transplantation.

Example

Example 1: Isolation of purified IgG

The present inventors performed four independent experiments purifying IgG from plasma according to the method of the present invention.

In all four experiments, fresh plasma was diluted 1:2 in phosphate-acetate buffer (1 part plasma to 2 parts buffer), the pH of the diluted plasma was about 4.6 to about 5.0, and the conductivity was 8 to 12 mS/cm. It was. The diluted plasma was transferred to a tank (suspension tank).

Octanoic acid was added to the diluted plasma in the suspension tank over a period of 20 to 60 minutes to dilute the buffer to an amount of at least 0.35 g/g total protein and mixed vigorously to create a plasma/octanoic acid emulsion. The emulsion was further incubated at approximately 22°C for 60 to 240 minutes. Without wishing to be bound by theory, it is believed that the temperature, slow addition, and vigorous mixing contribute to thorough distribution of octanoic acid throughout the diluted plasma. This is thought to result in more effective mixing of octanoic acid and albumin, and thus more albumin comes into contact with octanoic acid, leading to a higher yield of precipitate. The end result is a more effective separation of soluble immunoglobulins from insoluble albumin-octanoic acid complexes.

The resulting suspension is then fed into a continuous extraction filter unit, from which the residue is recycled back to the suspension tank, from which the filtrate (containing immunoglobulins) is fed into a second tank. The filtrate collected in the second tank was fed into a second device containing ultrafiltration and diafiltration systems. The retentate of the UF/DF system flowed back to the second tank, while the filtrate flowed back to the first tank.

The solution was diluted to a protein concentration of 20 g/L and the pH was adjusted to approximately pH 4.0 in the presence of polysorbate 80 (P80). The solution was subjected to depth filtration to further purify it. The resulting filtrate (referred to herein as “clarified filtrate”) was further evaluated to determine various product attributes as described further below.

The results demonstrate that the method of the present invention provides a highly efficient approach for isolating high levels of IgG directly from plasma samples. The yield of IgG is high and the recovery of IgG is consistent with other commercial manufacturing processes for isolating IgG. Immunoglobulin levels were measured by immunoturbidimetry.

The distribution of IgG subclasses was measured by immunoturbidimetry, and the results are shown in Figure 1, showing that the distribution of IgG subclasses in purified immunoglobulin preparations is similar to that found in plasma.

Example 2: Determination of protease and other contamination in IgG preparations

The concentration and extent of protease activity in the clarified filtrate were measured using standard methods. Briefly, a chromogenic substrate-based assay was used. Serine protease activity was measured by the cleavage ability of the chromogenic substrate Ile-Pro-Arg-pNA (S-2288) of the protein residue. During this reaction p-nitroaniline (pNA) is released, which is measured photometrically at 405 nm. Serine protease activity was measured at 37°C and pH 8.4. Kallikrein-like activity was measured by cleavage of the chromogenic substrate H-D-Pro-Phe-Arg-pNA (S-2302). During this reaction p-nitroaniline (pNA) is released, which is measured in kinetic mode using a photometer at 405 nm.

As shown in Figure 2, the method of the present invention effectively removes protease contamination.

Other impurities such as IgA, IgM, alpha1-antitrypsin were measured using standard techniques. Impurities such as IgA, IgM and ceruloplasmin were found to be present at low but still detectable levels. Other impurities (α-1-antitrypsin, α-2-macroglobulin, haptoglobin, hemopexin, fibrinogen, fibronectin, cholestrin, transferrin, triglycerides, and phospholipids) were found to be below detectable limits. The results are shown in Figure 3. Protein impurities were measured by immunoturbidimetry, while cholestrin triglyceride and phospholipid levels were measured using enzymatic assays.

The amounts of pre-kallikrein activator (PKA), factor IX and factor XI(a) were also measured. Their results are shown in Figure 4. PKA was measured with a chromogenic substrate (as described above), FIX was measured by ELISA, and FXI(a) was measured using the activated partial thromboplastin time (aPTT) assay.

The amount of albumin (g/L) in the starting plasma material was compared to the amount of albumin present in the IgG preparation (i.e., clarified filtrate) described in Example 1. The total albumin present in plasma was approximately 32.2 g/L. The amount of albumin in the IgG preparation of Example 1 was less than 0.341 g/L, which is consistent with the typical amount of albumin found in commercial grade IgG preparations. The results are shown in Figure 5.

Additional results shown in Figure 6 show that the immunoglobulin preparation generated in Example 1 has low levels of contaminating alpha/beta-globulin and albumin and is very rich in gammaglobulin. Gammaglobulin and alpha-/beta-globulin levels were measured using cellulose acetate/agarose electrophoresis.

Example 3: Isolation of purified albumin

After Example 1 was completed, the remaining suspension in the first tank was mixed with phosphate buffer (pH 7.1 to 7.4) to adjust the pH to 6.4 to 6.7. The resulting solution contained soluble albumin. Alternatively, after completing Example 1, the remaining suspension in the first tank was adjusted to a pH of 6.4 to 7.2 (preferably 6.8 to 7.2) using 1M sodium hydroxide. A combination of 0.12M sodium hydroxide and phosphate buffer is also considered.

The solution was further processed to achieve a total protein concentration of about 0.2 g/L to less than 1.0 g/L (or about 0.2 g/L to about 0.5 g/L).

Briefly, the albumin solution is fed to a continuous extraction filter unit, from which the residue is recycled back to the tank, from which the filtrate (containing albumin) is fed to a second tank. The filtrate collected in the second tank was fed to a second device comprising an ultrafiltration and diafiltration system. The retentate of the UF/DF system flowed back to the second tank, while the filtrate flowed back to the first tank.

Once the desired protein concentration was reached, the remainder of the UF/DF containing concentrated albumin was completed. This sample is referred to as “crude albumin after CE” in the table below.

Example 4: Evaluation of parameters

A number of independent experiments were performed to determine the optimal parameters for purifying IgG or albumin from plasma according to the method of the present invention.

For all experiments, fresh plasma was diluted 1:3 in phosphate-acetate buffer (1 part plasma to 2 parts buffer), and the diluted plasma had a pH of about 4.6 to about 5.0 and a conductivity of 8 to 12 mS/cm.

Laboratory scale experiments were performed to evaluate the effect of parameters such as ionic strength, pH, dilution ratio and type of diluent (and/or the effect of diluted acetic acid on impurity removal, recovery of IgG and albumin at different OA concentrations).

Yield and impurities at constant ionic strength and varying pH and OA amount

1kg of pooled plasma was diluted with 100mM sodium acetate, pH 4.0 (ratio: 1:3). The diluted plasma was divided into three equal portions and the pH was adjusted to the desired pH (4.2, 4.5 and 4.8) by dropwise addition of concentrated acetic acid with thorough mixing. Total protein concentration was determined by measuring absorption at A280. The conductivity of each aliquot was adjusted to within the range of 8.5+/-10mS/cm using acetate buffer.

Octanoic acid was added to the diluted plasma over a period of 20 to 40 minutes to dilute the buffer to an amount of at least 0.35 g/g total protein and mixed vigorously to create a plasma/octanoic acid emulsion, with a final concentration of 0.5 each. , 0.75 or 1.0 g/g total protein. The emulsion was further stirred for 60 to 180 minutes and then incubated with Cellpure 100 in a solution of 5 g/kg for 15 minutes. Then, filtration was performed using a CH9 filter layer.

Subsequent washes were performed at 20% of the starting volume using dilution buffer preadjusted to the same pH and conductivity with which the experiment was performed.

The clarified protein solution was then ultrafiltered up to 15-20 g/L. The pH of the solution was adjusted to 4.00±0.20 during diafiltration. The protein solution was then incubated at 37°C for 9 ± 1 h, and then the pH was shifted to 5.80 ± 0.10. Following a subsequent depth filtration step, the solution was loaded onto a strong anion exchange column, the purified IgG was collected as a fluid, and the pH was adjusted to 4.80±0.10.

result

At low pH (4.2) and low octanoic acid concentration (0.5 g/g protein), almost all albumin precipitates compared to pH 4.8 (Figure 8). At pH 4.8, significant amounts of albumin are still present in the clarified and diafiltered protein solution after treatment with octanoic acid. IgG yield was higher at pH 4.8 compared to pH 4.2 (Figure 7).

Low conductivity and high octanoic acid

The effect of low conductivity (2 to 5 mS/cm) at high octanoic acid concentrations (e.g., 1.0 g/g protein) at pH 4.20 was investigated.

The precipitation ability of octanoic acid at a concentration of 1 g/g protein and pH of 4.2 was investigated at various ionic strengths of 3, 4, 5 and 6.5 mS/cm.

result

The results (Table 6 and Figure 9) clearly show that for low conductivity (≤5 mS/cm) the IgG yield is lower compared to high conductivity (≥6 mS/cm). The albumin content of the clarified concentrated octanoic acid filtrate is similar to that at high conductivity.

High octanoic acid and different pH

Octanoic acid at a concentration of 0.55 g/g protein was investigated at different pH (4.2, 4.5 and 4.8) and different ionic strengths (5, 6, 7 and 8 mS/cm). See Tables 7 to 10 and Figures 10 to 13 below.

result

The data show that regardless of the octanoic acid concentration, the IgG yield at pH 4.8 is higher than at pH 4.2 and 4.5 (Table 7 and Figure 10). The residual amounts of albumin (Table 8 and Figure 11), IgA (Table 9 and Figure 12) and IgM (Table 10 and Figure 13) in the clarified and concentrated IgG solution are relatively low, and the addition downstream of the octanoic acid step It can be easily removed during process steps (e.g., major chromatographic purification). Therefore, the IgG solution can be sequentially further purified in the current process.

At pH 4.2, especially for albumin precipitation, the yield of IgG at high conductivity was still very acceptable, although slightly lower than at pH 4.8. Additionally, it is clear from the data that at appropriate octanoic acid concentrations (e.g., in the range of 0.50 to 0.55 g/g protein) a significant amount of albumin can be precipitated by octanoic acid so that albumin can be recovered in high yield and purity.

Example 5: Continuous filtration

In this experiment, the starting plasma pool was diluted in acetate or phosphate/acetate buffer with various ionic strengths (60mM, 80mM or 100mM) and pH values (4.0, 4.2, 4.5, 4.8 or 5.0).

One portion of plasma was diluted with two portions of buffer using an impeller mixer.

The diluted plasma was transferred to a tank (suspension tank). Octanoic acid was added to the diluted plasma in the suspension tank over a period of 20 to 40 minutes to bring the diluted plasma to amounts of 0.50 g OA/g protein, 0.75 OA/g protein, and 1.00 g OA/g protein, and heated vigorously. Mixing creates a plasma/octanoic acid emulsion.

The resulting suspension is then fed to a continuous extraction filter unit, from which the residue is recycled back to the suspension tank, from which the filtrate (containing immunoglobulins) is fed to the second tank. The filtrate collected in the second tank was fed to a second device comprising an ultrafiltration and diafiltration system. The retentate of the UF/DF system flowed back to the second tank, while the filtrate flowed back to the first tank.

The transmembrane pressure (TMP) is adjusted to ensure that the permeate (filtrate) flowing from the second unit is the same as the filtrate flowing from the first unit, ensuring a constant volume of the first tank during the extraction process.

The filtration unit is stopped when the protein concentration of the filtrate falls below a defined threshold and reaches a final dilution ratio of ≥1:X.

The dilution ratio (1:X) can be varied depending on the initial protein concentration of the plasma pool, the amount of OA used, and the desired protein concentration threshold of the residual OA suspension to be reached. Most average final dilution ratios are ≥1:20. In certain embodiments, the final dilution ratio was ≧1:12 and ≧1:15 or higher (e.g., 1:≧30).

Once the protein concentration reaches about 25 to about 30 g/L, the concentration step is complete (i.e., UF concentrate). During the final concentration the permeate flows to the waste.

Then, diafiltration begins. The concentrated protein solution was then diafiltered for a 10

The diafiltered solution was diluted to a protein concentration of 20 ± 2 g/L, and the pH was adjusted to approximately pH 4.0 ± 0.2. The solution was subjected to further purification depth filtration. The resulting filtrate (referred to herein as “clarified filtrate”) was further evaluated to determine various product attributes as further described in Example 6.

Example 6: Continuous filtration

2.6 L of cryo-enriched plasma was diluted using 1.26 L of 0.2 M acetic acid to pH 4.8 and conductivity 7.5 mS/cm.

Octanoic acid (0.447 g/g protein) was added slowly over a 60 minute time period with vigorous stirring to form an octanoic acid suspension with pH 4.76 and conductivity 7.72. The octanoic acid suspension was incubated at 20° C. for 3.25 hours and then transferred to the feed tank of the continuous extraction system of Example 1. The continuous extraction system was treated with 100mM sodium acetate buffer (pH 4.8) and recharged like the backflash tank.

The octanoic acid suspension was recirculated for a period of 15 minutes without filtration and then filtration was started. The backflash time was 15 seconds and the filtration time was 5 minutes. After about 5 minutes, the TFF system (example system shown in Figure 16) was started and recirculation was performed for about 4 hours as described above.

Once the protein concentration in the feed tank reached 0.2 to 0.5 g/L, the permeate was drained from the TFF system to waste.

The solution was diluted to a protein concentration of 20 g/L and the pH was adjusted to approximately pH 4.0 in the presence of polysorbate 80 (P80). The solution was subjected to further purification depth filtration.

50 mg of DEAE A-50/g protein was added to the clarified filtered solution, the pH was adjusted to 5.8 using Tris base, the solution was stirred for 60 minutes, and then the protein solution was loaded into a strong anion exchanger. The fluid was collected and the pH was adjusted to 4.8 using 0.2M HCl. The resulting fluid was further evaluated to determine various product properties as described in detail below.

All other product-related impurities were below the limit of quantification (see Figure 14).

Example 7: Additional experiments exploring parameters

In a series of experiments (hereinafter referred to as Examples 9 to 15), several parameters affecting the formation of insoluble albumin-octanoic acid complexes were investigated. The parameters are pH, ionic strength, OA concentration, dilution index and total recycle volume (final dilution ratio). These parameters more effectively separate soluble immunoglobulins from insoluble albumin-octanoic acid complexes.

The following table (Table 12) contains the experimental test conditions.

The results show excellent agreement with laboratory scale experiments. Table 13 shows the IgG yield and major impurities and residual albumin content in the clarified and concentrated OA filtrate.

The data shows consistent IgG yield with an average of 88.6% (range: 84.2 to 93.0%). The average residual albumin in the clarified solutions is less than 0.2 g of albumin per liter of plasma, except for Example 15. This is possible due to the high IgM content and low OA concentration of the plasma pool.

After recovery of the soluble protein-containing components (immunoglobulin G and other components such as IgA and IgM), all residual suspension and/or the first residue can be further processed to obtain purified albumin.

Example 8: Further processing of albumin

The residual suspension in the first retentate tank contains insoluble albumin-OA complex. The residual suspension contains less than 0.00035 g/L IgG, less than 0.0002 g/L IgA and less than 0.0002 g/L IgM.

To disrupt the binding between albumin and OA, the pH of the remaining suspension was adjusted to 6.4 to 6.7, preferably 6.8 to 7.2, using 1M sodium hydroxide. After mixing, the pH of the solubilized albumin is stable (30 to 60 minutes). The solution is fed to a continuous extraction system to produce an albumin-depleted retentate and an albumin-enriched filtrate. The filtrate is continuously concentrated up to 20-45 g/L protein using a TFF membrane (an exemplary system is shown in Figure 16).

The concentrated albumin solution is then heated at a temperature ranging from 60 to 65° C. over a time period of 90 minutes.

After adjusting the pH of the solution to 4.20 using 1M hydrochloric acid, the concentrated albumin solution was cooled to 4°C. A precipitate is formed, which dissolves during the breakdown of the albumin-OA complex at the mentioned pH.

The precipitate was removed by filtration. The protein in the filtrate consists mainly of albumin (purity >98% at 90% albumin yield). Table 14 and Figure 15 show the impurity profile of heat treated albumin.

It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual characteristic features mentioned or apparent from the text or drawings. All of these different combinations constitute various alternative embodiments of the invention.

Claims (78)

A method of obtaining a solution of immunoglobulin, preferably immunoglobulin G (IgG), comprising:
- contacting the blood-derived plasma sample with medium chain fatty acids under conditions capable of selectively precipitating albumin from the blood-derived plasma sample,
- the conditions are a pH range of about 4.6 to about 5.0,
- A method of obtaining an immunoglobulin solution, wherein the immunoglobulin solution is formed by said contacting step. A method for purifying immunoglobulins, comprising:
- contacting the blood-derived plasma sample with medium chain fatty acids under conditions of a pH ranging from about 4.6 to about 5.0 to allow albumin to selectively precipitate from the blood-derived plasma sample,
- thereby forming a suspension comprising a soluble protein-containing component comprising immunoglobulins and an insoluble protein-containing component comprising albumin, and
- A method for purifying immunoglobulins, comprising the step of separating the soluble protein-containing component from the insoluble protein-containing component to obtain a purified immunoglobulin solution. 3. The method of claim 2, wherein separating the soluble protein-containing component from the insoluble protein-containing component comprises feeding the suspension to a filtration unit, wherein the filtration unit separates the first retentate and the first filtrate. A method for purifying immunoglobulins, comprising a dynamic filter element configured to produce. The method of claim 3, further comprising recovering the first filtrate. 5. The method of claim 4, further comprising concentrating said first filtrate using a filtration unit optionally comprising dynamic filter elements or tangential flow filtration (TFF). A method for purifying immunoglobulins from plasma, comprising:
a) In a first tank, the blood-derived plasma sample is mixed with medium chain fatty acids under conditions of pH ranging from about 4.6 to about 5.0 to allow albumin to selectively precipitate from the blood-derived plasma sample, thereby producing soluble immunosorbent. forming a suspension comprising globulin and insoluble albumin,
b) feeding the suspension to a first filtration unit, wherein the first filtration unit is configured to produce a first retentate comprising the insoluble albumin and a first filtrate comprising the soluble immunoglobulin. The supply step, including,
c) optionally streaming the residue into the first tank to dilute the suspension in the first tank,
d) recovering the first filtrate from a second tank, and
e) optionally concentrating said first filtrate. A method of obtaining an immunoglobulin solution and albumin precipitate, comprising:
- contacting the blood-derived plasma sample with medium chain fatty acids under conditions allowing albumin to selectively precipitate from the blood-derived plasma sample,
- the conditions are a pH range of about 4.2 to about 5.0,
- A method of obtaining an immunoglobulin solution and an albumin precipitate, whereby an immunoglobulin solution and an albumin precipitate are formed. A method for purifying immunoglobulins and albumin, comprising:
- contacting the blood-derived plasma sample with a medium chain fatty acid under conditions of pH ranging from about 4.2 to about 5.0 to allow selective precipitation of albumin from the blood-derived plasma sample,
- thereby forming a suspension comprising a soluble protein-containing component comprising immunoglobulins and an insoluble protein-containing component comprising albumin, and
- A method for purifying immunoglobulins and albumin, comprising the step of separating the soluble protein-containing component from the insoluble protein-containing component to obtain a purified immunoglobulin solution and a suspension comprising albumin. 9. The method of claim 8, wherein separating the soluble protein-containing component from the insoluble protein-containing component comprises feeding the suspension to a filtration unit, wherein the filtration unit separates the first retentate and the first filtrate. A method for purifying immunoglobulins and albumin, comprising a dynamic filter element configured to produce. The method of claim 9, further comprising recovering the first filtrate. 11. The method of claim 10, further comprising concentrating said first filtrate using a filtration unit optionally comprising a dynamic filter element. A method for purifying immunoglobulins and albumin from plasma, comprising:
a) In a first tank, the blood-derived plasma sample is placed under conditions of a pH ranging from about 4.2 to about 5.0 and optionally a conductivity of from about 8 to about 12 mS/cm to allow selective precipitation of albumin from the blood-derived plasma sample. mixing the derived plasma sample and medium chain fatty acids to form a suspension comprising soluble immunoglobulins and insoluble albumin;
b) feeding the suspension to a first filtration unit, wherein the first filtration unit is configured to produce a first retentate comprising the insoluble albumin and a first filtrate comprising the soluble immunoglobulin. The supply step, including,
c) optionally streaming the first residue into the first tank to dilute the suspension in the first tank,
d) recovering the first filtrate from a second tank, and
e) optionally concentrating said first filtrate. As a method of obtaining albumin precipitate,
- contacting the blood-derived plasma sample with medium chain fatty acids under conditions allowing albumin to selectively precipitate from the blood-derived plasma sample,
- the conditions are a pH range of about 4.15 to about 4.25,
- A method of obtaining an albumin precipitate, whereby an albumin precipitate is formed. As a method for purifying albumin,
- contacting the blood-derived plasma sample with medium chain fatty acids under conditions of a pH ranging from about 4.15 to about 4.25 to allow albumin to selectively precipitate from the blood-derived plasma sample,
- thereby forming a suspension comprising a soluble protein-containing component comprising immunoglobulins and an insoluble protein-containing component comprising albumin, and
- A method for purifying albumin, comprising the step of separating the soluble protein-containing component from the insoluble protein-containing component to obtain a precipitate or suspension containing albumin. 15. The method of claim 14, wherein separating the soluble protein-containing component from the insoluble protein-containing component comprises feeding the suspension to a filtration unit, wherein the filtration unit separates the first retentate and the first filtrate. A method of purifying albumin, comprising a dynamic filter element configured to produce. The method of claim 15, further comprising recovering the first filtrate. 16. The method of claim 15, optionally further comprising concentrating said first filtrate using a filtration unit comprising a dynamic filter element. A method for purifying albumin from plasma, comprising:
a) In a first tank, the blood-derived plasma sample is placed under conditions of a pH ranging from about 4.15 to about 4.25, optionally with a conductivity of about 8 to about 12 mS/cm, to allow selective precipitation of albumin from the blood-derived plasma sample. mixing the derived plasma sample and medium chain fatty acids to form a suspension comprising soluble immunoglobulins and insoluble albumin;
b) feeding the suspension to a first filtration unit, wherein the first filtration unit is configured to produce a first retentate comprising the insoluble albumin and a first filtrate comprising the soluble immunoglobulin. The supply step, including,
c) optionally streaming the first residue into the first tank to dilute the suspension in the first tank,
d) recovering said first residue. 13. The method of claim 6 or 12, wherein step e) of concentrating the first filtrate comprises subjecting the filtrate to a continuous concentration process in a second filtration unit, wherein the immunoglobulin A method comprising static tangential flow filtration (TFF) or a dynamic filter element configured to produce a second filtrate enriched in second retentate and depleted in immunoglobulins. 20. The method of claim 19, wherein the immunoglobulin-depleted second filtrate is streamed back to the first tank. 21. The method of claim 19 or 20, wherein the immunoglobulin-enriched second residue is streamed back to the second tank. 22. The process according to any one of claims 19 to 21, wherein the dynamic filter element of the first filtration unit is configured to produce a filtrate (permeate) rich in soluble proteins (immunoglobulins). ) filter element, method. 23. The method according to any one of claims 19 to 22, wherein the dynamic filter element of the second filtration unit configured to produce the immunoglobulin-rich retentate is a dynamic cross-flow filter element or tangential flow filtration (TFF). . 24. A method according to claim 22 or 23, wherein the dynamic cross-flow filter element is a rotating cross-flow filter element. 25. A method according to claim 24, wherein the rotating cross filter element comprises a filter disk, preferably wherein the filter disk is mounted on a shaft member. 26. The method of claim 24 or 25, wherein the rotating cross filter element comprises at least one filter disk and at least one shaft member. 27. The method of claim 26, wherein the filter disk membrane is a ceramic membrane. 28. The method of any one of claims 1-27, wherein the plasma sample is from human blood. 29. The method of any one of claims 1 to 28, wherein the blood-derived plasma sample is fresh plasma, cryo-poor plasma or cryo-rich, optionally obtained from pooled plasma. ) A method comprising plasma, optionally the plasma being subjected to centrifugation. 30. The method of any one of claims 1 to 29, wherein the plasma sample does not include a filtering aid and/or has not been subjected to an alcohol fractionation process or another fractionation process. 7. The method of any one of claims 1-6, wherein the pH of the plasma sample is adjusted to a pH range of about 4.6 to about 5.0 prior to contacting with the medium chain fatty acid. 32. The method of claim 31, wherein the pH of the plasma sample is adjusted to a pH of about 4.6, about 4.7, about 4.8, about 4.9, or about 5.0. 13. The method of any one of claims 7-12, wherein the pH of the plasma sample is adjusted to a pH range of about 4.2 to about 5.0 prior to contacting with medium chain fatty acids. 34. The method of claim 33, wherein the pH of the plasma sample is adjusted to a pH of about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, or about 5.0. 19. The method of any one of claims 13-18, wherein the pH of the plasma sample is adjusted to a pH range of about 4.15 to about 4.25 prior to contacting with the medium chain fatty acid. 36. The method of claim 35, wherein the pH of the plasma sample is adjusted to a pH of about 4.15, about 4.2, or about 4.25. 37. The method according to any one of claims 1 to 36, wherein the plasma sample has a conductivity of from about 5 to about 12 mS/cm, preferably from 8 to about 12 mS/cm. 38. The method of claim 1 or 37, wherein the pH of the plasma sample is adjusted without substantially diluting the plasma prior to contacting the medium chain fatty acid. 38. The method of any one of claims 1-37, wherein the plasma sample is diluted in buffer before mixing with the medium chain fatty acids. 40. The method of claim 39, wherein the dilution ratio is about 1:0.5, about 1:0.75, about 1:1, about 1:1.25, about 1:1.5, about 1:1.75, about 1:2, about 1:3, or about 1:4 people, method. 41. The method according to any one of claims 1 to 40, wherein the medium chain fatty acid is selected from fatty acids with the formula CH ₃ (CH ₂ ) _n COOH, and the fatty acid is a C6 to C12 carboxylic acid. 42. The method of claim 41, wherein the fatty acid is selected from enanthic (heptanoic) acid, caprylic (octanoic) acid, pelargonic (nonano) acid or capric (decanoic) acid, or salts or esters thereof. 43. The method of claim 42, wherein the fatty acid is caprylic (octane) acid or a salt or ester thereof. 7. The method of any one of claims 1 to 6, wherein the amount of fatty acid, preferably caprylic (octanoic) acid, mixed with said plasma sample is about 0.30 g/g total protein (in plasma sample), about 0.35 g. /g total protein, about 0.40 g/g total protein, about 0.45 g/g total protein, or about 0.50 g/g total protein. 45. The method of claim 44, wherein the amount of fatty acid, preferably caprylic (octanoic) acid, is about 0.300 g/g total protein, about 0.325 g/g total protein, about 0.350 g/g total protein, about 0.375 g/g total. protein, about 0.400 g/g total protein, about 0.425 g/g total protein, or about 0.450 g/g total protein, and preferably the amount of said fatty acid, more preferably caprylic (octanoic) acid, is at least about 0.350 g. /g total protein, method. 13. The method according to any one of claims 7 to 12, wherein the amount of fatty acid, preferably caprylic (octanoic) acid, mixed with said plasma sample is about 0.35 g/g total protein (in plasma sample), about 0.40 g. /g total protein, about 0.45 g/g total protein, about 0.50 g/g total protein, or about 0.55 g/g total protein. 19. The method of any one of claims 13 to 18, wherein the amount of fatty acids, preferably caprylic (octanoic) acid, mixed with the plasma sample is at least about 0.35 g/g total protein. 48. The method of claim 47, wherein the amount of fatty acid, preferably caprylic (octanoic) acid, mixed with the plasma sample is greater than or equal to about 0.35 g/g total protein and less than 1.1 g/g total protein or about 1.1 g/g total protein. less than, how. 49. The method of any one of claims 1 to 48, wherein contacting the plasma sample with the medium chain fatty acid comprises mixing the plasma sample and the medium chain fatty acid to obtain a homogeneous emulsion of the medium chain fatty acid and the plasma sample. and preferably vigorous mixing to enable said mixing to form a homogeneous emulsion. 50. The method of any one of claims 1 to 49, wherein the plasma sample and the medium chain fatty acid, preferably caprylic (octanoic) acid, are separated for at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes. , mixing for a period of at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, or at least 60 minutes. 51. The method of any one of claims 1 to 50, wherein after the mixing step and before separating the soluble protein-containing component (soluble immunoglobulin) from the insoluble protein-containing component (insoluble albumin), an incubation period is followed. How to become. 52. The method of claim 51, wherein the incubation period is at least about 20 minutes, at least about 30 minutes, at least about 40 minutes, at least about 50 minutes, at least about 60 minutes, at least about 70 minutes, at least about 80 minutes, at least about 90 minutes, A method that is at least about 100 minutes, at least about 110 minutes, at least about 120 minutes, at least about 130 minutes, at least about 140 minutes, at least about 150 minutes or longer. 53. The method of any one of claims 1 to 52, wherein the steps of the method are performed at a temperature of about 18 to about 37°C, preferably about 18 to about 24°C. 54. The method according to any one of claims 1 to 53, wherein the purified immunoglobulin comprises immunoglobulin G (IgG), preferably human immunoglobulin G (IgG). 55. The method of any one of claims 1 to 54, wherein the immunoglobulin solution or concentrated immunoglobulin is further processed to further purify the immunoglobulin. 56. The method of claim 55, wherein the further processing does not include an additional continuous filter extraction step. 57. The method of claim 56, wherein the immunoglobulin is subjected to further processing, such as low pH treatment, chromatography steps (including anion exchange chromatography and/or immunoaffinity chromatography), virus filtration and inactivation steps, concentration and A method that is formulated to produce a final product that can be administered, for example, to humans. 58. The method of any one of claims 1 to 57, wherein the purified immunoglobulin solution contains impurities including IgA, IgM, albumin, α(alpha)-2 macroglobulin, α(alpha)-1 antitrypsin, lipids and lipoproteins. A method containing one or more of the following: 42. The method of any one of claims 1 to 41, wherein the insoluble protein-containing component of the suspension (i.e., obtained by contacting a plasma sample with a medium chain fatty acid) is separated from the insoluble protein-containing component. The method is held after the separating step. According to claim 6 or 12,
f) adjusting the pH of the insoluble protein-containing component comprising albumin to a pH of about 6.4 to 7.2 to obtain solubilized albumin,
g) optionally subjecting the solubilized albumin to further processing steps to remove impurities therefrom, and
h) the method further comprising recovering purified albumin from the solubilized albumin. 61. The method of claim 60, wherein the insoluble albumin is residual insoluble protein remaining in the first tank and/or the insoluble albumin may further comprise the first residue. 62. The method of claim 60 or 61, wherein the pH of the insoluble protein-containing component is adjusted without substantial dilution of the insoluble protein-containing component. 63. The method of any one of claims 60-62, wherein the conductivity of the insoluble protein-containing component is adjusted to about 8 to about 15 mS/cm. 61. The method of claim 60, wherein step f) first forms a further suspension by contacting the insoluble protein-containing component (insoluble albumin) with a buffer solution having a pH of 7.1 to 7.4 and optionally a conductivity of from about 8 to about 15 mS/cm. and adjusting the pH of the additional suspension to at least about 6.4, preferably to neutral pH, more preferably to a pH of about 6.4 to about 7.2 to obtain solubilized albumin. 65. The method of any one of claims 60-64, wherein the solubilized albumin is subjected to further processing steps to remove impurities. 65. The method of claim 65, wherein the method
- feeding the solubilized albumin to a filtration unit, wherein the filtration unit comprises a dynamic filter element configured to produce an albumin-depleted retentate and a filtrate rich in soluble albumin, and
- A method comprising recovering the albumin-rich filtrate. 65. The method of claim 65, wherein the method
i) providing the solubilized albumin to a first tank,
j) feeding the solubilized albumin solution to a first filtration unit, wherein the first filtration unit comprises a dynamic filter element configured to produce an albumin-depleted retentate and an albumin-enriched filtrate. ,
k) optionally streaming the residue into the first tank to dilute the solution in the first tank,
l) recovering the albumin-rich filtrate from a second tank, and
m) optionally concentrating the filtrate. 68. The method of claim 67, wherein step m) comprises subjecting the filtrate to a continuous concentration process in a second filtration unit, wherein the second filtration unit separates the albumin-enriched retentate and the albumin-depleted filtrate. A method comprising a dynamic filter element configured to generate. 69. The method of claim 67 or 68, wherein the albumin-depleted filtrate is streamed back to the first tank. 69. The method of any one of claims 67-69, wherein the albumin-rich retentate is streamed back to the second tank. 71. The method of any one of claims 67-70, wherein the dynamic filter element of the first filtration unit configured to produce an albumin-rich filtrate (permeate) is a dynamic crossflow filter element. 72. The method of any one of claims 67-71, wherein the dynamic filter element of the second filtration unit configured to concentrate the albumin solution to produce an albumin-rich retentate is a dynamic cross-flow filter element or TFF. 19. Method according to claim 18, wherein the pH of the first retentate is adjusted to 6.4 to 7.2, preferably 6.8 to 7.2, to solubilize the albumin. 74. The method of claim 73, wherein the solubilized albumin is fed to an additional filtration unit comprising a dynamic filter element to produce an albumin-depleted retentate and an albumin-enriched filtrate. 75. The method of claim 74, wherein the albumin-rich solution is heated in the range of 60 to 65°C. 76. The method of claim 75, wherein the heating occurs for a period of time greater than 90 minutes. 77. The method of claim 76, wherein after said heating step, the pH of the solution is adjusted to 4.20 or about 4.20 and preferably simultaneously cooled to 4° C. to allow protein denatured during said heating step to precipitate. 78. The method of claim 77, wherein by removing the precipitate by filtration, the filtrate produces purified albumin, preferably at least 95% total protein, at least 96% total protein, at least 97% total protein, or at least 98% total protein. A method comprising albumin purity and albumin yield of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, or at least 90%.